University of Southern California Dissertations and Theses

Immunotherapy of cancer

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Immunotherapy of cancer

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Content Copyright 2021
Aida Kouhi

IMMUNOTHERAPY OF CANCER

By

AIDA KOUHI

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2021
ii
ACKNOWLEDGEMENTS

My graduate program at USC has been an outstanding experience that has provided me
with an exceptional training, and I want to use this opportunity to thank those who helped me
complete my dissertation.
First and foremost, I would like to express my gratitude to my advisor Dr. Alan Epstein
for providing me with a rare privilege to join his team. The past a few years has been the best
years of my life because I have been able to participate in immunotherapy research, while
learning from experts in our group. Throughout the years, Dr. Epstein has not only allowed me to
mature as a scientist, but also taught me beyond the realm of science. I can’t imagine completing
my PhD without his mentorship and I am forever in his debt for all he has done for me.
I would like to deeply thank my thesis committee member Dr. Curtis Okamoto who has
been my mentor since the beginning of my graduate studies and has played a significant role in
my progress in the program. I would also like to thank Dr. Harvey Kaslow for his role as my
committee member and my mentor who has generously dedicated much of his time to ensure my
projects advance efficiently. I would also like to acknowledge with much gratitude other
members of Dr. Epstein’s team who have contributed to the completion of my projects: Dr.
Peisheng Hu, Dr. Long Zheng, and Vyshnavi Pachipulusu have had a significant contribution to
my projects. I would also like to thank Dr. Leslie Khawli, Luquin Ren, Tiffany Jehng for helping
me with my studies.
I would like to express my gratitude to Dr. Hossein Jadvar and Peter Conti for providing
me with an opportunity to join USC as a research assistant over a decade ago, which helped me
enter my graduate studies. I would like to thank Dr. Steven Swenson for always helping me with
my projects.
iii
I would also like to acknowledge the funding support for the projects I have included in
my thesis. My work in Dr. Epstein’s lab is supported by CellBT, Inc. The anti-ELP mAb
development was done with the direct supervision of Dr. Andrew MacKay, and this work was
supported by RO1 GM114839 and R01 EY026635 to JAM, P30 EY029220 to the USC
Ophthalmology Core Grant in Vision Research, P30 CA014089 to the USC Norris
Comprehensive Cancer Center, P30 DK048522 to the Liver Histology Core of the USC Research
Center for Liver Diseases, the USC Ming Hsieh Institute, the L.K. Whittier foundation, the
Gavin S. Herbert Endowed Chair of Pharmaceutical Sciences, the USC Nano Biophysics Core
Facility, the Translational Research Laboratory at USC School of Pharmacy, and the USC Cell
and Tissue Imaging Core.
Lastly, I would like to thank my family and friends for their unconditional love and
support during the most challenging years of my life; I would like to dedicate my thesis to my
parents who were my first science teachers and are a constant source of inspiration to me. I want
to thank my brother Ali, and my soul sisters Talayeh and Ava for always being there for me
whenever I needed them. I want to thank my best friend Golnar for her continued support of my
training, and Dr. Nazanin Entesari for her support and encouragement.

iv
Table of Contents
ACKNOWLEDGEMENTS ........................................................................................................... ii
LIST OF FIGURES .................................................................................................................... vi
LIST OF TABLES ..................................................................................................................... vii
ABSTRACT ............................................................................................................................. viii
Chapter One: Immunotherapy of Ovarian Cancer .................................................................... 1
1.1 Introduction ........................................................................................................................ 1
1.1.1. Ovarian Cancer .......................................................................................................................................... 1
1.1.2. CAR-T Cell therapy of Solid Tumors .......................................................................................................... 3
1.1.3. The human epidermal growth factor receptor 2 ...................................................................................... 4
1.2 Material and Method ................................................................................................................. 6
1.2.1. Mice .......................................................................................................................................................... 6
1.2.2. Reagents ................................................................................................................................................... 6
1.2.3. Cells ........................................................................................................................................................... 7
1.2.4. Binding Studies .......................................................................................................................................... 7
1.2.5. Vector Construction and Preparation of Lentivirus .................................................................................. 8
1.2.6. Primary T cell isolation, transduction, expansion, and analysis ................................................................ 9
1.2.7. In vitro functional Study .......................................................................................................................... 11
1.2.8. In Vivo Studies ......................................................................................................................................... 12
1.3 Results ..................................................................................................................................... 13
1.3.1. Selection of anti-HER2 antibody ............................................................................................................. 13
1.3.2. Expression of anti-HER2 CAR on Primary Human T cells ......................................................................... 14
1.3.3. In vitro functional Study .......................................................................................................................... 15
1.3.4. Anti-HER2 CAR T cells Eradicate SK-OV-3/ Luc-GFP Tumors in NSG Mice ............................................... 17
1.4 Discussion ................................................................................................................................ 18
1.5 References ............................................................................................................................... 21
1.6 Figures ..................................................................................................................................... 24
Chapter Two: Generation of a monoclonal antibody to detect Elastin-Like Polypeptides ....... 31
2.1 Introduction ...................................................................................................................... 31
2.2 Materials and Methods ............................................................................................................ 32
2.2.1. Cell culture and transfection .............................................................................................................. 32
2.2.2 Antigen (ELP) preparation ................................................................................................................... 33
2.2.3. Immunization Protocol ....................................................................................................................... 34
2.2.4. Generation of hybridomas .................................................................................................................. 35
2.2.5. Enzyme Linked Immunosorbent Assay (ELISA) ................................................................................... 36
2.2.6. Western Blotting ................................................................................................................................. 37
2.2.7. Immunofluorescence staining and confocal microscopy .................................................................... 38
2.2.8. Phase separation behavior of ELPs in presence of anti-ELP AK1 ........................................................ 39
2.3 Results ............................................................................................................................... 39
2.3.1. Expression, purification and characterization of antigens (ELPs) ....................................................... 39
2.3.2. Antibody production ........................................................................................................................... 41
2.3.3. Immunoreactivity of purified anti-ELP AK1, in an indirect ELISA ........................................................ 42
2.3.4. Western blot analysis of anti-ELP AK1 specificity ............................................................................... 43
v
2.3.5. AK1 Immunofluorescence staining of ELP-fusion proteins expressed in mammalian cells ................ 45
2.3.6. Phase separation of ELPs in presence of anti-ELP AK1 ....................................................................... 45
2.4 Discussion ......................................................................................................................... 47
2.6 References ......................................................................................................................... 51
2.7 Figures ............................................................................................................................... 53
2.8 Tables ................................................................................................................................ 65
COMPLETE LIST OF REFERENCES ............................................................................................ 66

vi
LIST OF FIGURES

Figure 1. Saturable binding curves for 10H8 and 8H11 anti-Her2 mAbs on SK-OV-3/ Luc-GFP
cells ............................................................................................................................................... 24
Figure 2. Binding patterns of anti-HER2 mAb on SK-OV-3/ Luc-GFP, OVCAR8/ Luc-GFP, and
Raji/ Luc-GFP cells. ...................................................................................................................... 25
Figure 3. Schematic representation of anti-Her2 CAR constructs and CAR T production and
expansion. ..................................................................................................................................... 26
Figure 4. Efficient expression of anti-Her2 10H8 CAR on T cells. ............................................. 27
Figure 5. Antigen-driven cytotoxicity of anti-Her2 CAR T cells. ................................................ 28
Figure 6. Antigen-driven release of cytokines from anti-Her2 CAR T cells. ............................... 29
Figure 7. Anti-Her2 CAR T cells eradicate SK-OV-3/Luc-GFP tumors in vivo in a metastatic
model of ovarian cancer. ............................................................................................................... 30
Figure 1. Purity and identity of ELPs used in immunization cocktail and/or characterization
assays ............................................................................................................................................ 53
Figure 2. Aliphatic ELPs stain poorly using Coomassie blue on SDS-PAGE. ............................ 54
Figure 3. Phase behavior for ELPs used in combination antigen immunization .......................... 55
Figure 4. Indirect ELISA of serum titer against purified ELPs was used to assess the combination
immunization of mice ................................................................................................................... 56
Figure 5. Purified anti-ELP AK1 detects a panel of ELPs using indirect ELISA ........................ 57
Figure 6. AK1 detects ELPs but not BSA during western immunoblotting. ................................ 58
Figure 7. AK1 efficiently detects ELPs during western immunoblotting from cellular lysates. .. 59
Figure 8. Immunofluorescence microscopy using anti-ELP AK1 detects temperature-dependent
assembly of ELP fusions in mammalian cells. ............................................................................. 60
Figure 9. Immunofluorescence microscopy using anti-ELP AK1 detects ELP fusions in
mammalian cells, but does not cross react with other proteins in untransfected cells. ................. 61
Figure 10. An optimal ratio of anti-ELP AK1 suppresses the ELP temperature-dependent shift in
optical density (OD). ..................................................................................................................... 62
Figure 11. NK-1 antibody of IgM isotype does not suppress the ELP temperature-dependent shift
in optical density (OD) .................................................................................................................. 63
Figure 12. Plasmid map of ELP constructs inside pet25b(+)backbone. ....................................... 64

vii
LIST OF TABLES

Table 2.1 Library of ELPs and ELP fusion proteins used in this study. 66

viii
ABSTRACT

The first chapter of this dissertation includes work on development of an immunotherapy
for treatment of ovarian cancer. Anti- HER2 CAR T-cells were developed to treat ovarian cancer
solid tumors that express HER2. CAR constructs were generated by fusing anti- HER2 scFv to a
second-generation CAR. The CAR vector was then fused with a lentivirus vector in-frame with
the CAR backbone and was used to transduce primary human CD3 positive T-cells. After
transduction, expanded CAR-T cells were characterized to confirm their ability to bind HER-2
antigen on HER-2 positive SK-OV-3 cells using flow cytometry. Expanded CAR-T cells were
then tested for their epitope-driven cytotoxicity by co-culturing them with HER-2 positive tumor
cells. In this assay, HER-2 CAR T-cells displayed dose-dependent cytotoxicity when co-cultured
with antigen positive but not antigen negative tumor cells. HER2 CAR T-cells were then tested in
vivo in an in-house intraperitoneal, metastatic model of human ovarian cancer that is used for
testing the efficacy of CAR-T cells against intraperitoneal tumors. Using this SK-OV-3/NSG
mouse model, HER-2 CAR T-cells were found to reduce in a significant manner tumor burden,
while tumor progression continued in mice that were treated with the controls. Results of this study
show that HER-2 CAR T cells have antigen driven effector functions both in vivo and in vitro and
can complement the current standard of care as a targeted therapy to improve the prognosis of
HER2 expressing ovarian cancer patients. In order to translate this product to the clinic, the murine
HER-2 antibody is presently being out-sourced for humanization.
The second chapter, which is a study that is published
*
focuses on development of
antibodies against an emerging class of peptide biologics is known as the Elastin-like polypeptide
(ELP). The identification and use of antibodies dominate the biologic, clinical diagnostic, and
therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein
ix
analytical experiments and to study the disposition of biologic therapeutics. ELPs are repetitive
protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of
ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during
expression. To facilitate these studies, we successfully generated a new mAb that is specific
towards ELPs and ELP fusion proteins. Purified antibody was evaluated in ELISA, Western
Blotting, and immunofluorescence assays for their analytical potential. The optimal anti-ELP mAb
proved highly reactive and specific towards ELPs. Moreover, these novel antibodies were able to
detect ELPs with a variety of aliphatic guest residues. ELPs phase separate in response to heating.
When incubated at great excess to ELP, the anti-ELP mAb partially blocks phase separation. These
findings are direct evidence that these novel murine mAbs will enable purification, experimental
detection, and characterization of these useful biopolymers.

*
Kouhi, A.; Yao, Z.; Zheng, L.; Li, Z.; Hu, P.; Epstein, A.L.; MacKay, J.A. Generation of a
Monoclonal Antibody to Detect Elastin-like Polypeptides. Biomacromolecules 2019, 20, 2942-
2952, doi:10.1021/acs.biomac.9b00503.
1
Chapter One: Immunotherapy of Ovarian Cancer

1.1 Introduction

1.1.1. Ovarian Cancer

Ovarian cancer (OC) is the seventh most common cancer affecting women globally, and
leading cause of death from gynecologic malignancies.
1, 2
In 2018, there were 300000 new cases
diagnosed worldwide, and this number is expected to rise to 371000 by 2035. Similar to other
types of cancer, OC is not a single disease and can be differentiated depending on its origin and
histology. The standard of care depends on the histologic diagnosis and stage of cancer but
generally includes cytoreduction surgery, followed by treatment with platinum-taxane based
combination chemotherapy.
3

Enhanced surgical techniques and superior treatment options have greatly improved the
prognosis for most solid tumors, OC prognosis, however, has remained relatively constant in the
last a few decades.
4
Several factors have contributed to the absence of meaningful clinical
improvement in management of OC including frequency of late-stage diagnosis, high rates of
recurrent cancer that eventually becomes resistant to available therapies, and lack of targeted
therapies.
5, 6

7

The stage of cancer at the time of diagnosis greatly impacts patient’s survival rate and
prognosis.
8
For example, the relative 5-year survival rate for localized disease is approximately
90% compared with 17% for a late stage- metastatic OC, and the overall survival rate is 48.6%.
(SEER)
Early diagnosis of OC has remained challenging, contributing to high mortality rates for the
disease.
9, 10
In fact, 80% of patients are diagnosed when the cancer is considered late stage because
of lack of symptoms and inadequate diagnostic markers in earlier stage patients.
4, 11
At present,
cancer antigen-125 (CA-125) in combination with transvaginal ultrasound is routinely used to
2
diagnose OC. CA-125, however, is not specific to the disease and is detected in benign conditions
such as endometriosis; additionally it is not sensitive for early stage detection as approximately
half of these patients do not express it.
12, 13
Other serum biomarkers such as HE4 have been
investigated in the clinic for better detection of the disease, none of which have proved to be
specific and sensitive enough to be approved as a single biomarker for diagnosis of OC, and this
is in part due to the heterogenous nature of the disease.
12, 14
Additionally, OC does not behave
similar to other solid tumors in order to metastasize, mainly due to its anatomical location leading
to absence of symptoms until the cancer is considered late stage. For most other cancers to
metastasize, tumor cells migrate through the blood or lymph nodes, ovarian tumor cells however
metastasize within the peritoneal cavity without the need to gain access to the blood or lymph.
2

Unfortunately, while for most patients initial treatment leads to favorable results, 60-80%
of patients will eventually relapse and become completely resistant to subsequent therapy.
2, 15, 16

For decades, first-line of therapy for most OC patients remained the same regardless of the cancer
subtype and stage, and clinical trials mostly focused on effects of dose intensity, frequency of
treatment, route of administration, and similar treatment plans on outcomes. In recent years,
significant progress has been made with respect to understanding the unique biological aspect of
different OC subtypes, tumor origin and heterogeneity of disease. According to well established
data, 90% of OC are of epithelial origin, but tumors can rarely derive from sex-cord stromal or
germline cells.
2, 17, 18
Epithelial ovarian cancer (EOC) is further subtyped into high-grade serous,
low-grade serous, mucinous, endometrioid, and clear cell carcinoma, with the high-grade serous
being the most commonly occurring and aggressive subtype (63%) .
2, 19
Some EOC tumors don’t
fall into these categories and are classified as malignant transitional cell (Brenner), mixed types or
undifferentiated carcinoma.
2, 20
Studies have examined the genetic pattern of these tumor subtypes
3
and have highlighted their mutational profiles that can be used to rationally design targeted
therapies for treatment of this lethal disease.
21
Immunotherapy based strategies have become at the
center stage of cancer research and therapy, and have shifted cancer treatment paradigm over the
last a few decades. Their power lies in their ability to engage the immune system to rapidly and
effectively eradicate tumors, making them one of the most promising approaches for cancer
therapy. Of the immunotherapies that have been approved to date, Chimeric Antigen Receptor
(CAR) T cell therapies have been a successful platform for treatment of cancer.
22
Unlike other
cell or antibody therapies, CAR T-cells are unique in that they can be described as “living
therapeutics” in that the number of CAR T-cells expand as they interact and kill their target. While,
these products are not yet approved to treatment of solid tumors, of the 300 active CAR T cell
therapy clinical trials registered globally, many are focused on extending the efficacy that is
evident in CAR treatment of hematological malignancies to solid tumors.
1.1.2. CAR-T Cell therapy of Solid Tumors

CAR T cell therapies frequently result in complete remission in patients with untreatable
metastatic malignancies that have exhausted all other treatment options. In brief, CAR T cell
therapies are engineered autologous T cells of a tumor bearing patient that have been genetically
modified ex vivo to encode receptors that recognize a tumor-specific antigen. Once these
genetically modified T cells are transfused back to the patient, they recognize, bind and eradicate
tumor. CAR T cell therapy relies on endogenous cell killing mechanisms that have been evolved
to eradicate cells that have become malignant and exert their cytotoxicity through similar signaling
pathways used by Cytotoxic T-cells but in an MHC independent manner.
23
This major difference
is in part responsible for the remarkable efficacy that is achieved by this approach because it allows
targeting cancer cells that frequently downregulate their MHC expression in an attempt to evade
4
cytotoxic T cell killing. Additionally, since CAR T cells form an immunological synapse with the
target cell through a single chain variable fragment (scFv) derived from an antibody, they can be
engineered to recognize numerous neoantigens that have been discovered as potential targets on a
variety of tumors.
CAR T cell therapies have had an exceptional clinical progress for treatment of
hematological malignancies, but their progress for treating solid tumors have been challenging. A
major challenge in targeting solid tumors with CARs is that the tumor microenvironment (TME)
creates conditions that are unfavorable for the survival and function of CAR T cells. The
immunosuppressive and hostile environments of solid tumors have been investigated for decades,
and the wealth of information from these studies have allowed various strategies to be considered
in designing CAR T cells that are able to properly function in the TME. These strategies include
careful consideration of the signaling and costimulatory domains, utilizing co-targeting antigens
that are overexpressed in the TME, and treatment strategies such as preconditioning
chemotherapies that deplete cells such as regulatory T cells (Tregs) that may inhibit CAR T cells.
24

Another key hurdle in development of CARs for treatment of solid tumors is identification
of cell surface proteins that are specific to the cancer and are not expressed on normal tissues in
order to avoid off target cytotoxicity.
25
It is for this same reason that of the monoclonal antibodies
(mAbs) that have been approved in the United States for treatment of cancer, only a small number
are indicated for treatment of solid tumors.
26
Several genetic mutations have been associated with
OC tumor development and progression, from which the human epidermal growth factor receptor
2 (HER2, ErbB2) is mainly discussed here, and is a potential target antigen for development of
CAR T cell therapy for treatment of OC.
1.1.3. The human epidermal growth factor receptor 2

5
The human epidermal growth factor receptor 2 (HER2, ErbB2) is a member of human
epidermal growth factor family of receptors (EGFR) also known as erythroblastic leukemia viral
oncogenes (ErbB), which in addition to HER2 consists of HER1, HER3, and HER4. These are
type I, transmembrane tyrosine kinase receptors with an extracellular domain that can bind ligands,
a transmembrane domain, and an intracellular domain with tyrosine kinase catalytic activity.
27, 28

To date, several endogenous ligands have been identified that can bind HER1, HER3, and HER4
receptors, upon which receptors homo- or heterodimerize. Upon binding a ligand, receptors are
auto-phosphorylated within their intracellular domain’s tyrosine residues and subsequently initiate
downstream signaling pathways that are essential for carcinogenesis including cell proliferation,
differentiation, apoptosis, adhesion, and survival.
29

HER2 does not bind any known ligand directly, but once other receptors of the family bind
their ligand, it is recruited as a partner and participates in the heterodimerization.
29
Another
distinction between HER2 and other receptors in the family is that HER2 is constitutively active,
whereas the extracellular domain of the other HER receptors alternates between an active and
closed conformation depending on the presence of a ligand.
30
HER2 has been shown to be
overexpressed and is associated with poor prognosis in a variety of solid tumors including breast,
ovarian, lung, colon, stomach, bladder, uterine cervix, and head and neck.
27, 31
HER2
overexpression has been successfully targeted with anti-HER2 mAbs in breast cancer patients that
overexpress the receptor, and it is now approved as a first line of therapy because of its remarkable
improvement of patient outcomes.
32
HER2 is overexpressed in a subset of patients with ovarian
cancer.
33, 34
According to various studies, 20-30% of OC cancer patients have increased expression
of HER2, which leads to poor prognosis in patients with advanced EOC.
27
It is now widely
accepted that the heterogeneity of OC cancer subtypes needs to be considered in development of
6
newer therapies, and overexpression of HER2 in a subset of OC patients may provide an
opportunity for development of superior therapy for treatment of this lethal cancer. Here in we
have designed CAR T cells targeted at HER2, which have been shown to be upregulated on various
cancer cells including OC.
1.2 Material and Method

1.2.1. Mice

Female NSG
™
mice, 8 to 12 weeks of age were either purchased from Jackson Laboratories (Stock
Number 005557) or bred at the USC animal facility. All mice experiments were conducted
according to a protocol approved by the USC Animal Care and Use Committee (IACUC protocol#
20585).
1.2.2. Reagents

The following reagents were used in this study: RPMI (Genesee Scientific), DMEM (Genesee
Scientific), dialyzed fetal calf serum (dFCS) (Hyclone, Catalogue #SH30079.03), GlutaMAX
(ThermoFisher, Catalog#35050-061), Penicillin/Streptomycin (Corning, Catalog#30-002-CI),
non-essential amino acids (Genesee Scientific, Catalog#25-536), Click’s medium (SIGMA,
Catalog#C5572-500ML), EcoRI (NEB, Catalog#R3101M), MluI (NEB, Catalog#R3198L),
psPAX2 (Addgene, Catalog#12260), pMD2.G (Addgene, Catalog#12259), Xfect (Clontech,
Catalog#631418), EasySep Human T cell isolation Kit (STEMCELL, Catalog#19051), Lentiblast
(OZBiosciences, Catalog#LB01500), 24-well G-Rex plates (Wilson Wolf , Catalog#80240M),
Dynabeads human T-activator CD3/CD28 (ThermoFisher, Catalog#11131D), ImmunoCult human
CD3/CD28/CD2 T cell activator (STEM CELL, Catalog#10970), IL-2 ELISA kit (ThermoFisher,
Catalog#EH2IL2 ), and INF-gamma ELISA kit (ThermoFisher, Catalog#EHIFN), Mycoplasma
testing kit (ThermoFisher, Catalog#M7006), APC conjugated goat anti-mouse IgG(H+L)
7
secondary antibody (eBioscience, Cat # 17-4010-82), Recombinant Human IL-2 (Peprotech, Cat
# 200-02) Recombinant Human IFN-γ (Peprotech, Cat # 300-02) IL-2 mAb (eBioscience, Cat #
14-7029-81) and IFN gamma mAb (eBioscience, Cat # 16-7318-81); the secondary antibodies
used were IL-2 antibody, Biotin (eBioscience, Cat # 13-7028-81) and IFN gamma mAbs (4S.B3),
Biotin (eBioscience, Cat # 13-7319-81), Peroxidase-conjugated Streptavidin (Jackson Immuno
Research Labs, Cat # NC9705430), TMB Substrate (BioLegend, Cat # 421101); IL-7-Fc, IL- 15-
Fc, and Dylight 650 anti-261tag antibodies were developed and prepared in our laboratory.
1.2.3. Cells

SK-OV-3 cells were obtained from and were developed to express Luc/GFP in-house, Raji cell
line was purchased from American Type Culture Collection (ATCC). OVCAR8/ Luc-GFP cell
line was gifted by Dr. Carlotta Glackin (City of Hope). Raji/ Luc-GFP cell line was gifted by Dr.
Yvonne Y. Chen (University of Southern California). Established human cancer cells were
cultured in complete medium which contained RPMI 1640, supplemented with 10% FCS, 1%
GlutaMax, 1% non-essential amino acids, and 1% Penicillin/ Streptomycin. HEK-293 LTV cells
were used for virus production and were purchased from Cell Biolabs (Cell Biolabs, Catalog#
LTV-100). HEK-293 LTV cells were cultured in DMEM media, supplemented with 10% FBS,
1% GlutaMax, 1% non-essential amino acids, and 1% Penicillin/ Streptomycin. Primary human
T-cells were purchased from Zen-Bio (Catalog# SER-BC-SDS), and isolated and enriched cells
were cultured in T-cell medium, which contained 50% Click’s medium, 36% RPMI-1640, 10%
dFCS, 2% GlutaMax, 1% non-essential amino acids, 1% Penicillin/ Streptomycin, 50 ng/mL IL-
7-FC and 100 ng/mL IL-15-FC. All cells were shown to be mycoplasma-free using a mycoplasma
test kit.
1.2.4. Binding Studies

8
Two anti-HER2 mAbs, 10H8 and 8H11, which were developed previously were screened and their
binding pattern to the HER2 overexpressing SK-OV-3/ Luc-GFP cell line were determined using
flow cytometry. Cells were incubated with varying concentration of mAbs ranging from 1 pM to
2 μM for 1 hour at 4 °C, washed with 2% dFCS in PBS, and incubated with APC conjugated goat
anti-mouse IgG(H+L) secondary antibody for 1 hour at 4 °C. Cells were washed with 2% dFCS in
PBS and the mean fluorescence intensity (MFI) was measured by flow cytometry using an Attune
Focusing Cytometer (Life Technologies, Carlsbad, CA). The binding pattern of 10H8 anti-HER2
mAb on OVCAR8/ Luc-GFP and Raji/ Luc-GFP cell lines were also determined by incubating
cells with the mAb using the same protocol described above. MFI was analyzed and plotted using
a Flowjo and GraphPad Prism software.
1.2.5. Vector Construction and Preparation of Lentivirus

Second generation anti-HER2 CAR constructs were used in this study and were engineered
according to a previous established protocol.
35
In brief, the cDNAs were synthesized by Integrative
DNA technologies (IDT) and comprised of CD8α leader sequence, single chain variable fragment
(scFv) derived from 10H8 or 8H11 mAbs, with the heavy chain (VH) and light chain (VL)
separated by a DNA sequence encoding for a (GGGGS repeated 3 times), a CD8α hinge, a CD8α
transmembrane or a CD28 transmembrane domain followed by a costimulatory and intracellular
signaling domain. The costimulatory domains were either 41BB or CD28, and the signaling
domains were either a CD3ζ or a mutated CD3ζ. Additionally, a 10 amino acid epitope (261-tag)
derived from human placenta growth factor was included in the CAR construct following the scFv
sequence (AVPPQQWALS), which allowed efficient detection of CAR expression on T cells
using Dylight 650-conjugated anti-261tag mAb according to a previous protocol.
35
The CAR
construct with the mutated CD3ζ signaling domain had point mutations in the tyrosine residues of
9
the CD3ζ signaling domain to eliminate the CAR T cell’s effector function and was therefore used
as a negative control in all in vivo experiments. The CAR’s cDNA was inserted into pLVX-EF1α-
IRES-Zsgreen (Clonetech, Catalog#631982) between the EcoRI and MluI restriction sites. The
IRES-Zsgreen encoding region was removed prior to inserting the CAR cDNA into the vector. For
production of lentivirus, HEK-293LTV cells were transiently co-transfected with the CAR transfer
vector mixed with two packaging vectors, psPAX2 and pMD2.G. After 24 hours, cell supernatants
were collected, and new cell culture media were added to the cells. The collected supernatants
were filtered and concentrated using ultracentrifugation at 20000g for 2 hours at 4 °C. Supernatants
were discarded and pelleted virus were resuspended in PBS, supplemented with 1% BSA and 7%
trehalose and virus were stored at 4 °C overnight. The same virus collection procedure was
repeated 48 hours post transfection. The two virus samples that were collected 24- and 48-hours
post transfection were combined, aliquoted and stored at -80 °C for subsequent experiments. The
three anti-HER2 constructs will be referred to as 10H8- 41BB, mutated 10H8- 41BB, and 10H8-
CD28 throughout the study, which corresponds to their costimulatory domains of the anti-HER2
CARs.
1.2.6. Primary T cell isolation, transduction, expansion, and analysis

T cells were obtained from peripheral blood mononuclear cells (PBMCs) that were isolated from
human buffy coat cells (Zenbio Inc.) using Ficoll-Paque and EasySep Human T cell Isolation Kit.
Isolated cells were cultured in T cell media as previously mentioned. T cells were activated on day
0 using Dynabeads human T-activator CD3/CD28 and were cultured in T cell media. Four days
after activation, and once T cells reached their exponential growth phase, they were transduced
according to a previously established protocols.
35
In brief, the desired lentivirus vectors (10H8-
41BB, mutated 10H8- 41BB or 10H8-CD28), lentiblast, and 10 μM of 1M HEPES were added to
10
250 μL T cell media in a non-tissue culture treated 24-well plates. The viral mixture was then
centrifuged at 2000g for 1 hour at 20˚C. After centrifugation, 0.5 million T cells in 250 μL T cell
media were added to the viral mixture and centrifuged at 1300g for 45 minutes at 25˚C. For a
negative control, in addition to mutated 10H8- 41BB, “mock T cells” were prepared by including
a group in which T cell were treated precisely as described above but no lentivirus was added to
the cells. Transduced cells were incubated in 24-well plates overnight at 37˚C, media was changed
after 24 hours and cells were then transferred to G-Rex plates. Dynabeads human T-activator
CD3/CD28 were removed from the T cell culture using an EasySep™ magnet 4 days after
transduction and transduction efficiencies were determined using Dylight 650-conjugated anti-
261tag mAb according to a previous study.
35
Briefly, T cells were incubated with the Dylight 650-
conjugated anti-261tag mAb for 30 minutes, washed with 2% dFCS in PBS, and fluorescence
intensity was subsequently measured by flow cytometry using an Attune Focusing Cytometer and
analyzed on a Flowjo™ software. On the same day, the three anti-HER2 CAR T cell groups
(mutated 10H8- 41BB, 10H8-41BB, 10H8-CD28) were enriched by incubating them in a non-
tissue culture treated 6-well plates that were pre-coated with anti-261tag mAb overnight. The next
day, plates were obtained and blocked with 2% FCS in PBS for 30 minutes. CAR T cells were
incubated in plates for 1 hour at 37˚C, then unbound T cells were removed from the plates, and
CAR expressing T cells were subsequently collected by pipetting the media up and down for at
least 20 times. Mock T cells were not used in the enrichment step because of lack of CAR
expression. Collected CAR T cells were transferred back to the G-rex plates and along with mock
T cells were re-stimulated using ImmunoCult human CD3/CD28/CD2 T cell activator. The T cell
media of CAR T cells were changed at least once every 2 days. Mock and enriched CAR T cells
were used in the in vitro and in vivo studies 6 days after re-stimulation.
11
1.2.7. In vitro functional Study

The in vitro cytotoxicity of transduced mock and anti-HER2 CAR and mock T cells (effector cells)
were tested with SK-OV-3/Luc-GFP and OVCAR8/Luc-GFP cells (target cells) using a previously
established protocol.
35, 36
In brief, 6 days after the anti-HER2 CAR and mock T cells were re-
stimulated, the percentage of anti-HER2 CAR expressing T cells (positive CAR T cells) in each
group were determined using Dylight 650-conjugated anti-261tag mAb as described above. CAR
T cells were co-incubated with target cells at varying effector to target ratios ranging from 2:1 to
0.03:1 (ratios were 2:1, 1:1, 0.5:1, 0.25:1, 0.13:1, 0.06:1 and 0.03:1) in 96-well flat bottom plates.
The percentage of positive CAR T cells were considered when calculating the effector cell number,
and therefore the number of reported effector cells represents the number of positive CAR T cells
and for each group the total number of T cells in each well slightly varied. The total number of T
cells in the mock group matched that of the group with the lowest CAR expression, which would
in turn have the highest number of total T cells. The mock and anti-HER2 T cells were incubated
with 0.1 million target cells in T cell media without any cytokines for 24 hours at 37˚C. In order
to generate a standard curve, 0.2 million target cells were serially diluted and seeded in separate
wells of the same plates without any effector cells. Control cells included the exact number of
target cells in the experimental wells and were used to correct for target cell viability independent
of effector cell killing mechanisms. A luminescent firefly luciferase assay was used to determine
the luminescence signal from target cells. A standard curve was generated based on the signal
intensity from serially diluted standard cells and the standard curve was used to extrapolate the
target cell number in each well. The plate was then read at 450nm on BIOTEK Synergy HT plate
reader. The live cell number was calculated as described above and cell lysis percentage was
calculated using the following formula, and plotted on a GraphPad Prism software:
12

%Lysis = '
(#target in control−#target in effector)
#target in control
7∗100%

The supernatants were collected and diluted by a factor of 5 with 1% BSA in PBS before being
used in a sandwich enzyme-linked immunosorbent assay (ELISA) to check for secreted IL-2 and
IFN-γ levels. High protein binding plates were coated with 2 µg/ml of IL-2 and IFN- γ mAbs,
sealed with a parafilm and incubated at 4˚C overnight. Plates were obtained and washed with a
wash buffer that contained 0.5% Tween20 in PBS, then 100 µl of the diluted samples or standards
were added to the plates. Standards were recombinant Human IL-2 and recombinant Human IFN-
γ, which were added at various concentrations ranging from 0.03ng/ml to 200ng/ml. Once samples
were added to the plates, they were incubated for 2 hours at room temperature, washed and 100µl
of biotinylated rabbit anti-human IL-2 antibody, or biotinylated rabbit anti-human IFN gamma
mAb were subsequently added to the plates and incubated for 1 hour at room temperature. Plates
were washed and 100µl peroxidase-conjugated Streptavidin was added to each well and incubated
for 45 minutes at room temperature. 3,3´,5,5´-tetramentylbenzidine (TMB) substrate was added to
the wells, and plates were incubated for 15 minutes at room temperature. To stop the reaction, 2N
H2SO4 was added to the wells and absorbance was measured at 450 nm using a Synergy™ HT
microplate reader. A standard curve was generated based on the signal intensity of the wells that
included serially diluted standards, and this curve was used to extrapolate the cytokine levels in
each well. Data was then plotted on a GraphPad Prism software.
1.2.8. In Vivo Studies

All animal experiments were performed according to an approved protocol by the USC Animal
Care and Use Committee (IACUC protocol# 20585). Female NSG
™
mice, ten weeks of age were
13
injected intraperitoneally with two million, SK-OV-3/ Luc-GFP expressing cells in 150 μL PBS
using a 26-gauge syringe. Tumor burden was determined 4 days after injection of the target cells
by measuring luciferase activity with an IVIS optical bioluminescence imaging modality. On the
same day, mice were randomly assigned to receive either mock, mutated 10H8- 41BB, 10H8-
41BB, or 10H8-CD28 anti-HER2 CAR T cells. Before injection, percentage of positive CAR T
cells were determined in each group by using a Dylight 650-conjugated anti-261tag antibody as
describe above. 6 million positive 10H8 anti-HER2 CAR T cells and mock T cells were prepared
and injected to tumor bearing mice intraperitoneally using a 26-gauge syringe. The percentage of
positive CAR T cells were considered when calculating the 10H8 anti-HER2 CAR T cells and the
number of cells injected to the mice in the mock group matched that of the group with the highest
number of total T cells. Bioluminescence imaging was used to monitor the tumor progression for
45 days using an IVIS optimal system, at which point the tumor burden in the mock and mutated
10H8- 41BB treated control groups would require euthanizing the mice.
1.3 Results
1.3.1. Selection of anti-HER2 antibody

Two anti-HER2 mAbs, 10H8 and 8H1, which were previously developed were screened and their
binding pattern to the HER2 expressing SK-OV-3/ Luc-GFP cell line was investigated using flow
cytometry.
37, 38
Both the 10H8 and 8H11 mAbs displayed a saturable binding pattern on SK-OV-
3 cell when cells were incubated at various antibody concentrations ranging from 1 pM to 2 μM
and the ED50 for10H8 and 8H11were 4.2 and 3.9 nM respectively (Figure 1). The binding pattern
of 10H8 anti-HER2 mAb to OVCAR8/ Luc-GFP, and Raji/ Luc-GFP cells were also determined
and compared to that of the SK-OV-3/ Luc-GFP cells. Previous studies have shown that OVCAR8
cell line has lower HER-2 expression compared to the SK-OV-3 cell line and Raji cell line do not
express HER-2 receptor.
34
The results of this study suggested that the 10H8 anti-HER2 mAb may
14
have a lower binding to the OVCAR8/ Luc-GFP cells as compared to the SK-OV-/ Luc-GFP cells
because the mean fluorescence intensity (MFI) of the same 10H8 anti-HER2 mAb concentration
binding to the OVCAR8/ Luc-GFP cells was significantly lower compared to the SK-OV-/ Luc-
GFP cells (Figure 2). 10H8 anti-HER2 mAb showed no binding to the Raji/ Luc-GFP cells even
at the highest concentration of 2 μM (Figure 2).
1.3.2. Expression of anti-HER2 CAR on Primary Human T cells

To engineer a CAR vector against HER-2, second generation anti-HER2 CAR constructs were
engineered by inserting their CAR cDNA into a modified, self-inactivating pLVX-EF1α lentiviral
vector (Figure 3a). The scFvs were derived from either 10H8 or 8H11 anti-HER2 mAbs; The 261-
tag was included in the CAR construct to enable efficient analysis of CAR cell surface expression
on T cells using a Dylight 650 conjugated anti-261tag mAb.
39
The intracellular costimulatory
domains were either 41BB, or CD28 and the signaling domain were either CD3ζ or mutated CD3ζ.
The two different costimulatory domains, 41BB and CD28, were utilized to determine the most
effective construct for targeting HER-2 expressing tumors since the two function differently.
40
The
mutated CD3ζ signaling domain which was constructed so that it was unable to initiate a
downstream signaling pathway was included as a negative control. This construct was included in
the study to reveal any potential efficacy as a result of scFv’s interaction with the target antigen,
independent of the CAR T cell signaling. Mock T cells were used also as a negative control and
were prepared by subjecting T cells to identical transduction conditions as the anti-HER2 CAR T
cells except CAR lentivirus was not added to this group. This construct revealed in vivo and in
vitro cytotoxicity due to the allogenic effects because T cells and tumor cells were not from the
same donors. Once T cells were activated, they were transduced when they reached exponential
growth phase (Figure 3b). Four days after transduction, the transduction efficiencies of T cells
15
were determined by flow cytometry using anti-261 tag mAb. Production of the CAR lentivirus
were not successful for any of the 8H11 anti-HER2 CAR vectors since no CAR expression was
detected on transduced cells; therefore the 8H11 CARs were not used in subsequent experiments.
The transduction efficiency for the 10H8 anti-HER2 CARs ranged from 17% to 32% depending
on the CAR construct and mock T cells did not have any CAR expression since no CAR lentivirus
were added to these cells during transduction (Figure 4a). In order to enrich the percentage of T
cells that were positive for CAR expression in each group, T cells were incubated in plates that
were pre-coated with anti-261 tag mAb, unbound cells were removed, and bound cells, which
would be the CAR expressing T cell population were retrieved. The mock cells were not enriched
because they did not express the CAR construct and would therefore not bind to the anti-261 tag
mAb on the plates. CAR T cells were then transferred to G-Rex plates, and along with the mock
T cells they were re-stimulated using ImmunoCult human CD3/CD28/CD2 T cell activator
antibody. T cells were cultured in T cell media for use in subsequent experiments, and supernatant
were replaced once every two days. According to previously published studies from our lab, the
ImmunoCult human CD3/CD28/CD2 T cell activator antibody was superior to Dynabeads human
T-activator CD3/CD28 for re-stimulation because it would allow CD4+ and CD8+ T cell
population to expand equally, whereas lack of CD2 would lead to higher expansion of CD4+
population.
39
The percentage of T cells positive for CAR expression were determined 6 days after
re-stimulation, and expression level increased to 60% to 80%, which was significantly higher to
the levels before the enrichment for the three CAR expressing anti-HER 2 T cells (Figure 4b).
Finally, when the in vivo functional studies were performed, the in vitro studies were completed
in parallel.
1.3.3. In vitro functional Study

16
Antigen driven cytotoxicity of anti-HER2 10H8 CAR T cells
Cytolytic activity and effector function of mock and 10H8 anti-HER2 CAR T cells against tumor
cells were determined by co-incubating them with SK-OV-3/ Luc-GFP and OVCAR8/ Luc-GFP
target cells at various ratios. Twenty-four hours after co-incubation, percentage of lysed target
cells were determined by analyzing the luminescence intensity of target cells and extrapolating the
percentage of lysed cells using a standard curve and control cells. Both 10H8-41BB and 10H8-
CD28 anti-HER2 CAR T cells efficiently lysed SK-OV-3/ Luc-GFP cells and the percentage of
lysed cells decreased as the ratio of effector to target cells decreased (Figure 5a). The cytolytic
activity of both CAR T cells were similar regardless of their costimulatory domain (Figure 5a).
The efficiency of 10H8 anti-HER2 CAR T cells in lysing OVCAR8/ Luc-GFP cells was lower
compared to that of the SK-OV-3/ Luc-GFP cells (Figure 5b). Additionally, while both 10H8-
41BB and 10H8-CD28 CAR T cells exhibited equal cytolytic activity against SK-OV-3/ Luc-GFP
cells, their cytotoxicity against OVCAR8/ Luc-GFP cells varied based on the CARs costimulatory
domain. According to this study, the 10H8-CD28 CAR T cells displayed significantly higher
cytotoxicity against target cells compared with the 10H8-41BB CAR T cells. These data
demonstrate that the costimulatory domain of the CAR T cells results in different cytolytic activity
of effector cells depending on target antigen’s expression levels on target cells. These data are in
line with previous findings that CAR T cells comprised of CD28 costimulatory domains are more
sensitive to the target antigen’s expression levels on the target cells, meaning that even at lower
antigen expression levels, they will maintain efficient cytolytic activity.
40
The cytolytic activity of
mock T cells against both target cells were negligible for most of the effector to target ratios, and
even for the highest ratio, T cells lysed approximately less than 30% of target cells (Figure 5).
Antigen driven Release of Cytokines from anti-HER2 CAR T cells
17
Cytokine production by the mock and CAR T cells was assessed by measuring secreted levels of
IL-2 and IFN-γ in supernatant of effector and target cells that were co-incubated in the cytotoxicity
assay. While 10H8-41BB and 10H8-CD28 CAR T cells displayed equal in vitro cytotoxicity
against SK-OV-3/ Luc-GFP cells, cytokine production varied depending on the CARs
costimulatory domains (Figure 6a). Slightly lower levels of cytokines were secreted by the 10H8-
41BB effector cells when compared with that of the 10H8-CD28 effector cells. This difference
could be due to the functional differences between the two costimulatory domains that have been
studied and demonstrated extensively in the past.
40
In fact, previous studies have shown that
effector CAR T cells that were comprised of 41BB costimulatory domains secrete lower levels of
cytokines such as IL-2 compared with ones that are constructed with CD28.
40
Only 10H8-CD28
effector T cells secreted cytokines when cells were cocultured with OVCAR8 (Figure 6b). These
data were consistent with this construct having higher cytolytic efficiency when compared
with10H8-41BB T cells, and also previous findings that effector CAR T cells that are comprised
of 41BB costimulatory domains secrete lower cytokine levels than CD28 containing CARs.
40

1.3.4. Anti-HER2 CAR T cells Eradicate SK-OV-3/ Luc-GFP Tumors in NSG Mice

The efficiency of anti-HER2 10H8 CAR T cells at eradicating HER2 overexpressing SK-OV-3/
Luc-GFP cells were evaluated in NSG
™
mice that were injected with tumor cells interperitoneally.
Four days after injection of target cells, tumor burden was confirmed by bioluminescence imaging;
on the same day, mice were randomly assigned and received 6 million 10H8 anti-HER2 CAR or
mock T cells. The positive experimental groups were treated with 108H-41BB and 108H-CD28 T
cells and the negative control groups were treated with mutated 108H- 41BB and mock T cell.
Tumor progression was monitored for 40 days by bioluminescence imaging of tumor bearing mice
using an IVIS Spectrum Imaging system. Progression of tumor burden continued in the two groups
18
that were treated with mutated 10H8- 41BB or mock T cells (Figure 7). The tumor burden in mice
that were treated with either 10H8- 41BB or 10H8- CD28 T cells significantly decreased as evident
in the first imaging that was performed on day 7 weekly thereafter (Figure 7). The tumor burden
continued to decrease in the majority of mice and by day 40
th
, only one mouse in each of the 10H8-
41BB or 10H8- CD28 T cell treated groups did not respond to therapy. Additionally, a small tumor
burden was detected in 4 mice in the 10H8- 41BB CAR T cell treated group (mice# 2,6,8 and 10),
and one mouse in the 10H8- CD28 CAR T cell treated group (mouse# 8). The residual tumors that
was detected in these mice were located at the anatomical site where the intraperitoneal (IP)
injections were performed. We hypothesize that this occurred most likely when the needle was
withdrawn from the intraperitoneal space since prior wiping of the needles before injection did not
prevent tumor growth either in the subcutaneous or intradermal space. In conclusion, the
observations from these in vivo studies demonstrate that the 10H8-41BB and 10H8-CD28 anti-
HER2 CAR T cells have substantial in vivo efficacy and significantly decrease tumor burden in a
HER2 expressing metastatic model of ovarian cancer growing in the peritoneal cavity similar to
where these tumors grow and spread in women.
1.4 Discussion

Ovarian cancer (OC) is an aggressive and lethal cancer that is considered to be an area of
unmet medical need due to poor clinical outcomes that has failed to improve in the past three
decades. The majority of OC patients are diagnosed when the disease is considered late stage,
which results in unfavorable prognosis and low 5-year survival rates. OC is a heterogenous disease
with various subtypes, each of which have been shown to have distinct cellular origin and
histological features; but regardless of the cancer subtype, the first line of treatment for most
patients is removal of the tumor followed by treatment with a platinum-taxane based combination
19
chemotherapy. While, most patients respond to initial therapy, majority of them will experience a
relapse and become completely resistant to therapy. The first line of treatment for OC remained
unchanged until the last a few years, which targeted therapies started to emerge for targeting this
heterogenous disease owing to better understanding of its molecular basis. In this study, we
investigated the efficacy of anti HER-2 CAR T cell therapies in eradicating a HER2
overexpressing model of ovarian cancer. Our studies demonstrate that anti-HER2 CAR T cells that
were engineered and used in this study were able to effectively eradicate tumor in the in vivo and
in vitro studies. The anti-HER2 CARs that were engineered were 10H8-41BB and 10H8-CD28
CARs and were used to determine if two constructs exhibit different antitumoral activities based
on the functional differences of their costimulatory domains. While both CARs demonstrated
similar in vivo efficiency in lysing HER2 overexpressing SK-OV-3/ Luc-GFP target cells, 10H8-
CD28 CARs had higher efficiency in lysing OVCAR8/ Luc-GFP cells which have been shown to
have lower expression of target antigen. Therefore, the properties of CAR’s costimulatory domain
may play an important role in efficiency of CAR T cells’ cytolytic activity against tumor cells, and
together with target antigen’s expression levels needs to be considered in designing CAR T cells
with efficient cytolytic activity again target tumor cells. In contrast to the in vivo cytotoxicity
studies, both the 10H8-41BB and 10H8-CD28 CARs displayed similar antitumoral activities in
vivo, and the fictional differences between the two costimulatory domains did not result in different
in vivo efficacies. Both ani-HER2 CARs significantly reduced tumor burden in SK-OV-3/ Luc-
GFP bearing mice, while tumor progression continued in mice that were treated with the mock and
mutated 10H8- 41BB CAR T cell treated control groups. Results of this study demonstrate that
anti-HER2 CAR T cells have antigen driven effector functions in vivo and in vitro and can be
20
added to the standard of care as a targeted therapy and improve prognosis of HER2 expressing
ovarian cancer patients.

21
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24
1.6 Figures

Figure 1. Saturable binding curves for 10H8 and 8H11 anti-Her2 mAbs on SK-OV-3/ Luc-
GFP cells
SK-OV-3/Luc-GFP cells were incubated with various concentrations of 10H8 and 8H11 anti-
HER2 mAbs ranging from 1 pM to 2 mM and the Mean Fluorescence Intensity (MFI) was
measured by flow cytometry using an Attune Focusing Cytometer. Both 10H8 and 8H11 mAbs
showed saturable binding patterns on cells. ED50 for 10H8 and 8H11 were 4.2 and 3.9 nM,
respectively.
25

Figure 2. Binding patterns of anti-HER2 mAb on SK-OV-3/ Luc-GFP, OVCAR8/ Luc-
GFP, and Raji/ Luc-GFP cells.
Cells were incubated with various concentrations of mAbs ranging from 1 pM to 2 mM and the
mean fluorescence intensity (MFI) was measured by flow cytometry using an Attune Focusing
Cytometer. The lower MFI observed when 10H8 mAb was incubated with SK-OV-3/ Luc-GFP
cells compared to that of the OVCAR8/ Luc-GFP cells suggesting that there is lower binding
of 10H8 mAb to the OVCAR8/ Luc-GFP cells. The 10H8 mAb showed no binding to the Raji/
Luc-GFP cells even at the highest concentration.

26

Figure 3. Schematic representation of anti-Her2 CAR constructs and CAR T production
and expansion.
(a) Schematic representation of anti-Her2 CAR constructs which were comprised of a CD8α
leader sequence, single chain variable fragment (scFv) derived from the 10H8 mAb, with the
heavy chains (VH) and light chains (VL) separated by a linker, followed by a CD8α hinge, a
CD8α or CD 28 transmembrane domain, and intracellular costimulatory and signaling domains.
Additionally, a 10 amino acid epitope (261-tag) derived from human placenta growth factor
was included in the construct following the scFv sequence. (b) Schematic representation of the
production and expansion of CAR T cells. T cells were activated on day 0 and were transduced
on day 4. They were restimulated on day 9 and used for animal studies on day 15.

27

Figure 4. Efficient expression of anti-Her2 10H8 CAR on T cells.
(a) CAR expression was measured on mock and primary transduced T cells 5 days after
transduction by flow cytometry analysis using Dylight 650-conjugated antibody against 261
tag. The percentage of positive CAR T-cells for each of the constructs are shown above. (b)
CAR expression after enrichment for the above constructs is shown using Dylight 650-
conjugated antibody against 261 tag and flow cytometry. The percentage of CAR T cells
increased by these procedures as above.

28

Figure 5. Antigen-driven cytotoxicity of anti-Her2 CAR T cells.
(a) Cytotoxicity of mock, 10H8-41BB, and 10H8-CD28 against SK-OV-3/ Luc-GFP cells are
shown at increasing effector/target ratios. The 10H8-41BB and 10H8-CD28 CAR-T cells
showed 95.6% and 100% cell lysis, respectively, and the mock T cells showed 15.9% cell
lysis at effector/target cell ratios of 2:1. (b) Cytotoxicity of mock, 10H8-41BB, and 10H8-
CD28 against OVCAR8/ Luc-GFP cells is shown . The 10H8-41BB and 10H8-CD28 CAR-T
cells showed 70.3% and 98.5% cell lysis, respectively, and the mock T cells showed 33.6%
cell lysis at effector/target cell ratios of 2:1.

29

Figure 6. Antigen-driven release of cytokines from anti-Her2 CAR T cells.
(a) Quantitation of IL-2 and IFN-γ secreted by mock, 10H8-41BB, and 10H8-CD28 co-cultured
with SK-OV-3/ Luc-GFP cells at various effector/target ratios is shown. 10H8-41BB and 10H8-
CD28 produced 0.5ng/ml and 0.7ng/ml of IL-2, respectively, and 1ng/ml and 3ng/ml of IFN-γ,
respectively, at an effector/target ratio of 2:1. (b) Quantitation of IL-2 and IFN-γ secreted by
mock, 10H8-41BB, and 10H8-CD28 co-cultured with OVCAR8/ Luc-GFP cells at various
effector/target ratios is shown. 10H8-41BB and 10H8-CD28 produced 0ng/ml and 0.12ng/ml
of IL-2, respectively, and 0.2ng/ml and 1.45ng/ml of IFN-γ, respectively, at an effector/target
ratio of 2:1.

30

Figure 7. Anti-Her2 CAR T cells eradicate SK-OV-3/Luc-GFP tumors in vivo in a
metastatic model of ovarian cancer.
(a) Schematic diagram of the in vivo study. NSG mice were inoculated with SK-OV-3/ Luc-
GFP cells on day 0 and were treated with mock (n=5), mutated 10H8-41BB (n=5), 10H8-
41BB (n=10) and 10H8-CD28 (n=10) CAR T cells on day 4. (b) Bioluminescence images of
the mice on days 1, 6, 13, and 20 of therapy. The groups treated with 10H8-41BB and 10H8-
CD28 CAR T showed significant tumor reduction 6 days after treatment, but each had one
non-responsive to therapy. By contrast, the control groups (mock and mutated10H8 -41BB
CAR T treated groups) showed tumor progression for the entire duration of the study.

a
31
Chapter Two: Generation of a monoclonal antibody to detect Elastin-Like
Polypeptides

2.1 Introduction

Elastin-like polypeptides (ELPs) are a unique class of protein polymers whose sequence is derived
from human tropoelastin, the precursor to elastin. As an emerging class of biopolymers with a
broad range of applications, ELPs have been extensively studied in the last few decades. ELPs are
repeated pentameric motifs with the amino acid sequence [Val-Pro-Gly-Xaa-Gly]n. A function of
Xaa and n, ELPs display inverse phase transition temperatures, Tt, above which they phase
separate from aqueous solution. When ELPs are heated above Tt, they phase separate from bulk
solution into a secondary liquid phase called a coacervate. Coacervation is reversible and ELPs
become soluble when they are cooled below Tt. ELPs are monodisperse, biodegradable, and
biocompatible, which makes them versatile agents for in vitro and in vivo applications. ELPs may
be useful to generate therapeutics because they can: i) stabilize multivalent nanoparticles that
modulate cellular signaling;
1
ii) be genetically fused to antibodies without the need for
bioconjugation chemistry;
3
iii) potently engage multiple receptors;
4
iv) undergo proteolytic
biodegradation after endocytosis;
5
and v) and some ELP sequences have the potential for low
antigenicity due to derivation from human tropoelastin.
7

Despite these potential applications, no anti-ELP mAbs have yet been reported. A major
limitation in the clinical translation of ELP biologics has been a lack of mAbs to characterize their
identity during expression, and disposition upon cellular uptake. As mentioned above, the
identification and use of antibodies dominate the biologic, clinical diagnostic, and therapeutic
landscapes.
9, 10
In particular antibodies have become essential tools in a variety of protein
32
analytical experiments and can quantitate the amount and location of a biologic target. To the
best of our knowledge, anti-ELP mAbs are not commercially available and no other groups have
yet reported their successful production. Thus, the potential applications for ELPs would likely
increase with the identification of a mAb capable of binding ELPs specifically.
In addition to the above-mentioned applications, antibody-based assays are widely used
in early biologic development phases and become even more essential at the mid- and late-stage
discovery.
11
From a regulatory standpoint mAb reagents are often used in batch release testing,
and they have readily become a routine part of standard operating procedures.
11
Their critical
role in product development has made it difficult for biologics that lack a specific antibody against
them to complete the development phase. Aimed at facilitating ELP studies, we herein report
successful production of an anti-ELP mAb, designated AK1. Specifically, studies are presented to
show the reactivity of these new reagents in ELISA assays, Western Blotting, and protein
purification studies. Furthermore, this manuscript shows this antibody can be used in
immunofluorescence assays to determine subcellular localization. Immunofluorescence based
assays that were employed in this study elucidated that AK1 has the potential to not only detect
ELPs in antibody-based assays, but also more importantly reveal specific behavior of these
protein polymers such as their ability to coacervate and form punctate microdomains in vivo.
While polyclonal antibodies have long been used in research, this manuscript describes
development of a mAb against ELPs. The advantage of mAb generation is elimination of batch-
to-batch variability that is often observed with polyclonal antibodies.
2.2 Materials and Methods

2.2.1. Cell culture and transfection

33
The NS0 murine myeloma cell line was utilized in production of hybridomas. Cells were cultured
in RPMI 1640 medium containing 15% fetal calf serum, 100 U/ml Penicillin-G, and 100 µM/ml
streptomycin sulfate. 293T cells were cultured in DMEM containing 10% FBS. Lipofectamine 3000
was used for transient transfection of 293T cells according to manufacturer’s instructions. Cells
were grown in a humidified, 5% CO 2 incubator at 37 °C and hybridomas were frozen in liquid
nitrogen for long-term storage using cryostor (Biolife Solutions Inc, Bothel, WA). All cultures were
shown to be mycoplasma free by PCR mycoplasma test kit (PromoCell GmbH, Heidelberg,
Germany).
2.2.2 Antigen (ELP) preparation

This study employed various ELP libraries as listed in Table 1. Recombinant pET-25b(+) plasmids,
containing ELP genes, were previously engineered in our lab using recursive directional ligation
method.
6, 8, 12
BLR (DE3) Escherichia Coli competent cells (Novagen Inc., Milwaukee, WI) were
transformed with the ELP expression vectors. Transfected cells were streaked on an LB agar plate
and incubated overnight at 37 °C. A single colony was selected and inoculated in a 50 mL Terrific
Broth medium overnight with shaking at 37 °C. The starter culture was used to inoculate 1 L
Terrific Broth with shaking at 37 °C. All the aforementioned plates and liquid media contained
100 µg/ml of carbenicillin. Bacterial cultures were centrifuged, resuspended in cold phosphate
buffered saline (PBS) and lysed on ice using a probe-tip sonicator (Misonix, Farmingdales, NY).
The cell lysate was centrifuged at 17,000 g at 4 °C to remove insoluble debris. Polyethyleneimine
(0.5% w/v) was added to the crude cell lysate to precipitate nucleic acids, and after 20 minutes
of incubation on ice the solution was centrifuged. Soluble ELP proteins were purified from the
supernatant using an established technique called inverse transition cycling.
13, 14
Typically 4-5
34
rounds of heating/cooling were enough to attain a sufficient purity. The final purified protein
batches were filtered using a 0.2 µm polyethersulfone sterile membrane (Pall Corporation, Port
Washington, NY). Protein concentrations were determined by solubilizing purified proteins in an
8 M Guanidine-HCL solution (Thermo Fisher Scientific, Waltham, MA) and measuring their
absorbance at 280 nm using UV-Vis spectroscopy (ε ELP= 1,285 M
-1
cm
-1
) according to a previously
established method.
15
To assess protein purity, 5 µg of samples were resolved on a 4-20%
gradient Tris-Glycine-SDS PAGE gel (Lonza, Basel, Switzerland) stained by a 10% copper chloride
solution. This ELP library was unable to be stained by Coomassie HRP conjugated molecular
weight standard was also loaded onto the gel as a positive control (Bio-Rad Laboratories,
Hercules, CA). Matrix Assisted Laser Desorption Ion Time of flight mass spectrometry (MALDI-
TOF) was used to confirm the exact molecular weight of expressed constructs. ELP Tt was
determined according to a previously published method.
16
Briefly, for each ELP, varying
concentrations were prepared (5-100 µM) in PBS, and added to Tm microcell cuvettes (Beckman
Coulter, Brea, CA). Then temperature was increased 1 °C per minute, and optical density (OD) at
350 nm was recorded three times per minute (every 0.3 °C). The maximum first derivative of the
OD with respect to temperature was defined to be the Tt.
2.2.3. Immunization Protocol

Three ELPs antigens described previously were used as immunogens in this study.
6
An 1 ml PBS
aliquot containing 100 µg/ml murine GM-CSF and 10 µg of each of the three ELP antigens was
prepared, and emulsified in 1.5 ml of adjuvant using an 18-gauge micro-emulsifying needle. Five
BALB/c female mice, 8 weeks of age, were subcutaneously injected with 0.1-0.2 µl of inoculum
at several sites using a 22-gauge needle and a glass syringe once every two weeks for six weeks.
35
For the first two immunizations, the antigens were emulsified in complete Freund’s adjuvant
(MilliporeSigma, Burlington, MA), and for the third and subsequent immunizations using
incomplete adjuvant (MilliporeSigma, Burlington, MA). One week after the third or fourth
immunization, serum antibody titers were monitored using an indirect ELISA. Two mice with the
highest antibody titer received one final intravenous booster injection containing 10 µg of each
of the three ELP antigens dissolved in PBS. Four days later, the two mice that received the booster
injection were euthanized by cervical dislocation and their spleens were removed using aseptic
techniques.
2.2.4. Generation of hybridomas

For fusion, single splenocyte suspensions from the two mice that received the booster injection
were prepared and fused with 8-azaguanine-resistant mouse myeloma NS-0 cells at a ratio of 3:1
using 40% polyethylene glycol, MW 1450 (MilliporeSigma, Burlington, MA) as previously
described.
17
Fused hybridoma cells were seeded in 96-well flat bottom culture plates and
incubated in RPMI 1640 complete medium supplemented with 1X hypoxanthine-aminopterin-
thymidine (HAT) selection medium (MilliporeSigma, Burlington, MA). After two weeks, HAT
medium was replaced by HA medium (HAT medium lacking aminopterin) (MilliporeSigma,
Burlington, MA). Supernatants from wells with growing cells were screened for anti-ELP antibody
production using an indirect ELISA, and wells that were positive for all three ELPs were subcloned
by limiting dilution. Positive subclones were further screened and two hybridoma cell lines that
produced highly reactive mAbs (AK1, AK2) were selected for expansion and purification. A rapid
antibody isotyping kit (Thermo Fisher Scientific, Waltham, MA) was used to determine the
isotype of the anti-ELP AK1. In this manuscript, only AK1 was characterized.
36
2.2.5. Enzyme Linked Immunosorbent Assay (ELISA)

Indirect ELISA was used to monitor the relative serum antibody titer of immunized mice and to
screen for antibody secreting hybridomas. To assess serum reactivity, the three antigens used in
immunization of the mice were separately diluted in PBS. High protein binding 96-well plates
were separately coated with each of the three diluted antigens (100 µl/well, 2 µg/ml). Coated
plates were sealed to prevent evaporation and incubated overnight at 4 °C. Plates were washed
three times with phosphate buffered saline (pH 7) containing 0.5 % Tween-20 solution (PBST).
Free protein binding sites were then blocked with 1X casein blocking buffer (MilliporeSigma,
Burlington, MA) in PBS for one hour at 37 °C. Plates were incubated with serially diluted mouse
sera, or purified AK1 in the blocking buffer for one hour at 37 °C. Following three PBST washes,
plates were incubated with a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated goat
anti-mouse IgM (Jackson Immunoresearch Labs, West Grove, Pennsylvania) in blocking buffer for
one hour at 37 °C. Plates were washed again three times with PBST, and 100 µl of 3,3´,5,5´-
tetramentylbenzidine (TMB) substrate (MilliporeSigma, Burlington, MA) was added to each well,
and plates were incubated at room temperature for 15 minutes. To stop the reaction, 100 µl of
2N H 2SO 4 was added to each well and absorbance was measured at 450 nm using a Synergy™ HT
microplate reader (BioTek, Winooski, VT). The apparent equilibrium disassociation of binding
constant Kd was calculated using GraphPad Prism software; the statistical analysis was performed
by fitting the Abs450 ELISA curve with nonlinear regression to the following saturation binding
equation:
𝐴𝑏𝑠
!"# %&
=
'
!"#
(
!$%
)
&
*(
!$%
Eq. 1
37
Where CmAb is the concentration of the antibody, Bmax is the maximum shift in Abs450, nonspecific
binding was subtracted from the data set and background was constrained to a constant value
of zero. Nonspecific binding was determined by measuring mean absorbance of wells that were
coated with the antigen, followed by incubation with the secondary antibody.
2.2.6. Western Blotting

The reactivity of AK1 against antigens was further assessed in Western Blotting assays in which
anti-ELP AK1 was used to detect either pure ELPs, ELP fusion proteins in a cell lysate, or to track
ELPs during purification. To detect pure ELPs, a library of purified protein mixtures containing 1X
Laemmli sample buffer (Bio-Rad, Hercules, CA) were prepared and heated at 70 °C for 10 minutes.
Proteins were then separated on a PAGEr EX 4-12% gradient gel (Lonza, Morristown, NJ) and
transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA) using an
iBlot2 dry blotting system (Thermo Fisher Scientific, Waltham, MA). Membranes were
immunoblotted with anti-ELP AK1 (1:4000 dilution) at 4 °C overnight, washed, and incubated with
an HRP-conjugated goat anti-mouse IgM (Santa Cruz Biotechnology, Dallas, Texas). Membranes
were washed and a Super Signal™ West Pico Plus chemiluminescent substrate (Thermo Fisher
Scientific, Waltham, MA) was used to visualize protein bands. Membranes were imaged using a
ChemiDoc Touch Imaging system (Bio-Rad Laboratories, Hercules, CA). To blot ELP proteins from
whole cell lysates, 293T cells were seeded in 35 mm dishes and transfected with ELP fusion
proteins the following day. Forty-eight hours later, whole cell lysates were prepared using RIPA
buffer (Cell Signaling Technology, Danvers, MA). Total protein was loaded onto gels as described
above and the same protocol used for immunoblotting pure ELPs was followed. To detect and
track ELPs during purification, ELPs were expressed and purified as described above. During the
38
purification process, a sample from various steps of the process was collected and stored. The
following day, stored samples were diluted 10 times and 1.7 µl from each sample was loaded
onto the gel, and the same protocol used for immunoblotting pure ELPs was followed.
2.2.7. Immunofluorescence staining and confocal microscopy

Immunofluorescence assays were performed with ELP-transfected 293T cells to assess the
reactivity of purified AK1 against ELPs. Cells were cultured and plated on glass coverslips in 12-
well plates using serum free DMEM culture media and transfected with ELP fusion protein vectors
that were previously engineered by our team using lipofectamine 3000 (Thermo Fisher Scientist,
Waltham, MA).
8
Briefly, either A96 or V96 DNA sequences were fused at the N terminus of
clathrin-light chain (CLC) with a myc epitope in between. Thirty-six hours after transfection, cells
were incubated on ice for 45 min, followed by 45 min of incubation on ice or at 37 °C. Cells were
then fixed with 4% paraformaldehyde (Alfa Aesar, Haverhill, MA) in PBS, washed with 50 mM
ammonium chloride, and permeabilized with 0.1% Triton X-100 in PBS at room temperature.
Permeabilized cells were washed with PBS and blocked with 1% BSA in PBS for 1 hour at 37 °C.
Cells were then incubated with chicken anti-myc antibody (Abcam, Cambridge, MA) for 1 hour at
37 °C, washed, and incubated with Alexa Flour 633 conjugated goat anti-chicken secondary
antibody (Invitrogen, Carlsbad, CA) for another hour at 37 °C. After anti-myc staining, cells were
washed and incubated with anti-ELP AK1, washed and incubated with Alexa Flour 430 conjugated
goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at 37 °C. Cells were
washed and stained with DAPI and mounted with coverslips using fluoromount (Diagnostic
Biosystems, Pleasanton, CA). Confocal images were captured on a Zeiss LSM 800 confocal
microscope (Carl Zeiss Microscopy, Thornwood, NY).
39
2.2.8. Phase separation behavior of ELPs in presence of anti-ELP AK1

To measure ELP’s Tt in the presence of AK1, the following mixtures were prepared in PBS on ice.
A sample containing 1 µM V96 and no AK1; a sample containing 1 µM V96 and 0.33 µM AK1 ([3:1]
V96 to antibody molar ratio); a sample containing 1 µM V96 and 0.2 µM AK1 ([5:1] V96 to
antibody molar ratio); a sample containing 1 µM V96 and 0.1 µM AK1 ([10:1] V96 to antibody
molar ratio); and a sample containing 1 µM V96 and 0.02 µM AK1 ([50:1] V96 to antibody molar
ratio). Samples were mixed on ice and incubated at 4 °C for 30 min while shaking to promote
binding. Mixtures were then added to Tm microcell cuvettes (Beckman Coulter, Brea, CA). Then
temperature was increased 1 °C per minute, and OD at 350 nm was measured and recorded three
times per minute (every 0.3 °C). Co-incubation of ELP with AK1 did not significantly shift the Tt;
however, the major effect was elimination of the temperature-dependent increase in OD. To
quantify this effect in each cuvette, the Area Under the Curve (𝐴𝑈𝐶
+#,-"#℃
) was estimated as
follows:
𝐴𝑈𝐶
+#,-"#℃
= ∑
(0
'()
10
'
)(34+"#
'()
*34+"#
'
)
5
%
678
Eq. 2
Where the OD350 was baseline corrected to zero at T = 30 °C, and the trapezoidal rule was used
to sum up the AUC in increments, i, ranging from T = 30 °C until sample n, which occurred at T =
50 °C. Statistical comparison between groups was performed using a global ANOVA (alpha = 0.05)
followed by the Tukey post-hoc test between each group.
2.3 Results

2.3.1. Expression, purification and characterization of antigens (ELPs)

A small panel of ELP protein polymers were prepared, characterized and used as antigens during
the immunization step of antibody development (Table 1). Three ELPs were chosen to facilitate
40
identification of antibodies capable of binding a broad range of ELPs. A192 is a soluble protein
polymer, which is not expected to phase separate in vivo, and is generally referred to as a
monoblock ELP. In contrast, A96I96 and S48I48 are both diblock copolymers that form stable
nanoparticles at physiological temperatures.
6
For both diblock copolymers, the hydrophobic ELP
(Xaa=Ile) forms the hydrophobic core of the particle, and the hydrophilic ELP (Xaa=Ala, Xaa=Ser)
stabilizes the corona. Above the critical micelle temperature (CMT) for the hydrophobic block,
the hydrophilic corona sterically stabilizes nanoparticles in aqueous solutions. When compared
with S48I48, A96I96 forms larger particles (Rh= 40 vs. 24 nm) above slightly lower temperatures
(CMT = 21 vs. 27 C).
2, 18
Aside from the ELPs that were used in immunization, several other ELPs
were also prepared and used for in vitro studies to investigate whether AK1 can detect ELPs with
different aliphatic sequences. The ELPs examined cover four different guest residues, and various
ELP states (soluble, coacervate, nanoparticle) at physiological temperature. Vectors encoding the
desired ELP genes that were previously engineered by our team were transformed into E. coli
BLR (DE3) cells for expression.
2, 18
ELP proteins were purified from bacterial cell lysate using an
established method known as inverse transition cycling.
6, 19
Four to five rounds of heated/cooled
centrifugation yielded proteins with good purity, which were characterized using copper chloride
stained SDS-PAGE (>90%; Figure 8). Sensitivity of staining methods for protein visualization is an
important factor when determining protein purity upon purification. While there are studies
showing that copper chloride may be more sensitive than Coomassie blue staining in protein
visualization
20
, the amino acid sequence of the ELP protein-polymer is also an important factor
to be considered. For the ELPs in this study, copper chloride is a superior staining technique when
compared with Coomassie blue staining. Coomassie blue staining barely detects these ELPs on
41
SDS-PAGE and did not reveal significant impurities that might have been absent on copper
chloride stained gels (Figure 9). Therefore, the copper chloride staining was used for analysis of
protein purity and molecular weight. The exact molecular weight of expressed ELP constructs
was confirmed using MALDI-TOF (Table 1). This technique was also used to elucidate absence of
major protein contamination in purified ELP protein samples. Having confirmed the molecular
weight (MW) and purity of protein polymers, their phase separation behavior for the ELPs used
during immunization was confirmed by monitoring their optical density as a function of
temperature and concentration (Figure 10). As expected, A192 remained soluble below its Tt,
and phase separated above 55 °C. A96I96 and S48I48 are both referred to as diblock copolymers,
which displayed a biphasic change in optical density as temperature increased. The change in
optical density associated with a lower temperature is due to the formation of a stable
nanoparticle as the hydrophobic block (Xaa=Ile) phase separates and forms the core of
nanoparticles, also defined as CMT.
6
As the temperature further increases, the hydrophilic shells
(Xaa= Ser or Ala) of nanoparticles phase separate and allow formation of larger structures. The
higher temperature at which the hydrophilic blocks phase separate is called the bulk transition
temperature (T t,bulk).
6

2.3.2. Antibody production

With the goal of generating an antibody capable of binding multiple aliphatic ELP libraries, a
group of five BALB/c female mice were hyperimmunized with a small ELP library (Table 1). After
3-4 immunizations over a period of six-eight weeks, antibody titers were checked in the sera of
the mice using an indirect ELISA (Figure 11). This assay was employed to ensure that the mice
developed anti-ELP antibodies and the immunization protocol was sufficient for antibody
42
production. Mouse plasma showed the greatest immunoreactivity against A192 (Figure 11A).
Plasma from none of the mice was initially immunoreactive against the diblock copolymer A96I96
(Figure 11B); furthermore, only mouse #5 was reactive against S48I48 (Figure 11C). A96I96 and
S48I48 both form micelles at physiological temperature, while A192 remains soluble. These
structures were most likely maintained in the ELISA assay given the experiment was performed
at room temperature in the absence of denaturing agents. The observation that mice showed
immunoreactivity against the soluble ELP but not the nanoparticles was unexpected and remains
unexplained. The two mice with the highest serum antibody titers against A192 (mice # 2, and 5)
received one final booster injection and were used for further antibody production. Hybridoma
cell lines were developed by the fusion of antibody-secreting splenocytes of the mice that
received the booster injection with myeloma NS-0 cells. Hybridoma clones were screened for
reactivity against ELPs by using their supernatant in an indirect ELISA, and clones with the best
results were again subcloned by limiting dilution to isolate stable monoclonal cell lines. Subclones
were further screened using the same method, and two subclones (AK1 and AK2) with good
reactivity against all three ELP antigens were used for expansion, freezing, and future antibody
production. The resultant mAbs were determined to be of an IgM heavy chain subclass using a
rapid antibody isotyping kit. Having an approximate yield of 30 mg/L culture, AK1 was used in
subsequent assays for further antibody characterization.
2.3.3. Immunoreactivity of purified anti-ELP AK1, in an indirect ELISA

The reactivity of purified anti-ELP AK1 against a panel of ELPs (Table 1) including the three ELPs
used in the immunization step was tested in an ELISA assay. AK1 was serially diluted and added
to antigen coated multiwell plates (200 ng antigen/well). AK1 displayed a clear concentration
43
dependent reactivity against all ELPs that were tested even when it was diluted to low nM
concentrations (Figure 12). The apparent antibody binding constant (K d) was estimated for 5 ELP
antigens ranging from 0.4 to 0.6 nM. The only notable exception to this was that the K d for S96
was approximately one order of magnitude weaker, at 5 nM. As mentioned before, all ELPs
(including the 6 ELPs tested here) consist of a repeated pentapeptide with 4 other amino acids of
the pentapeptide being identical; therefore, the differences between these ELPs are confined to
a single amino acid and the number of times the pentapeptide is repeated. Of the 6 antigens
tested in the ELISA, only 3 of them were used in the immunization. This assay suggests that the
antibody-antigen interaction is relatively independent of the guest amino acid residue (Xaa) in
the repeated pentapeptide sequence of ELPs.
2.3.4. Western blot analysis of anti-ELP AK1 specificity

Specificity of AK1 against a library of ELPs was assessed using Western Blotting. AK1 was able to
detect not only the three ELPs that were used in immunization of the mice (Figure 13), but also
other ELPs, including ELP fusion proteins with two ELP guest residues (Figure 14). Anti-ELP AK1
showed strong reactivity with pure ELPs in Western Blot analysis (Figure 14A). Similar to the
results of the indirect ELISA, AK1 reacted with all ELPs that were tested in the assay regardless of
their guest amino acid residue. An unexpected observation in the Western Blot analysis was that
the minimum concentration of I48 that had to be loaded onto the gel for the antibody to be able
to react with the protein was higher than that of the other 5 purified ELPs (Figure 14A). For the
other 5 ELPs, 0.1 μg per lane was sufficient for the antibody to detect the protein bands. I48 ELP,
however, did not react with the antibody unless 2 μg per lane was loaded onto the gel. This
finding was different than the antibody-antigen reactivity that was observed in the indirect ELISA
44
assay mentioned above. In the indirect ELISA, I48 had similar reactivity when compared with the
other 5 ELPs. This difference could be due to the ELP-immobilization methods used in the
Western Blot vs. indirect ELISA procedures. AK1 also displayed specific reactivity with ELP-
containing fusion proteins in a crude cell lysate (Figure 14B). In this experiment, 293T cells were
transfected with ELP fusion proteins A96-CLC and V96-CLC. Both of these fusions include the
clathrin-light chain (CLC), and have been previously characterized by our group.
8
Transfected cells
were lysed, run on a gel, and evaluated by Western Blot. As seen on the blot, the antibody
specifically detected bands corresponding to the expected MW of ELP fusion proteins. The last
lane of the blot was loaded with a cell lysate from 293T cells that were not transfected with ELP
fusions. As expected, no bands were detected, which confirms that the antibody-antigen
interaction is specific to the ELP fusions. The ability of the AK1 to detect ELP and ELP fusion
proteins in Western Blotting analysis could be an invaluable tool for their purification and
analysis. As mentioned earlier, ELP-mediated phase separation is used for the purification of ELPs
during which cooling and heating cycles are used to isolate ELPs from other proteins in a cell
lysate. During the cooling cycle, ELP fusions are expected to become soluble and remain in the
supernatant when centrifuged. However, ELP fusions that are not folded correctly can aggregate
and form pellets when centrifuged after a cooling cycle. It has often been difficult to predict in
which step of the process one has lost the ELP fusions during purification. As seen in Figure 14C,
AK1 can be used to track ELPs in the bacterial lysate and also through two rounds of thermal
cycling, confirming that this approach can be used in future studies to better optimize ELP
expression and purification procedures.
45
2.3.5. AK1 Immunofluorescence staining of ELP-fusion proteins expressed in
mammalian cells

Secondary immunofluorescence was used to demonstrate the ability of anti-ELP AK1 to stain cells
expressing ELP fusion proteins, which were previously reported by our team.
8
293T cells were
transiently transfected with genes encoding ELPs (A96 and V96) fused to CLC. 2 days after
transfection, 293T cells expressing ELPs were incubated on ice for 45 minutes to allow ELP fusions
to solubilize. Cells were then incubated at either 4 or 37 °C after which they were first stained
with an anti-myc antibody and next by anti-ELP AK1. AK1 positively reacted with ELP fusion
expressing cells without any apparent cross-reactivity with untransfected cells (Figure 15).
Furthermore, anti-ELP AK1 stained V96-CLC transfected cells that were incubated at 4 °C
differently from the cells that were incubated at 37 °C. Consistent with previous reports from our
team, when V96-CLC transfected cells were incubated above the intracellular Tt, the fusions
assemble punctate microdomains.
8
In contrast, cells incubated at 4 °C, which is below the
intracellular Tt, retain a more diffuse, cytosolic staining pattern for anti-ELP AK1. As a control, the
A96-CLC fusion protein was not observed assembling microdomains at 37 °C, which is consistent
with its higher expected intracellular Tt (Table 1). Both above and below Tt, the anti-myc mAb
and anti-ELP AK1 had similar staining patterns. Untransfected cells do not stain with AK1 (Figure
16). These data clearly show that it is possible to directly stain intracellular ELP microdomains
without the need for additional epitope tags, such as myc.
2.3.6. Phase separation of ELPs in presence of anti-ELP AK1

As previously mentioned, ELPs are temperature sensitive polypeptides that undergo reversible
phase separation above an inverse phase transition temperature. It has previously been shown
that association of ELPs with other molecules in the microenvironment can change their phase
46
behavior;
4
furthermore, the ELP-mediated phase separation has been proposed as a way to
enhance the duration of delivery from an extravascular injection site.
21
To further examine this
concept, phase separation of V96 ELPs in presence of AK1 was observed by measuring OD at 350
nm as a function of temperature. The molar concentration of V96 was fixed at 1 μM, which
displays an ~ 37 °C Tt. This ELP sequence and concentration was selected to ensure that AK1
remains thermally stable near the temperature of the ELP-mediated phase separation. At the
molar ratio of V96 to AK1 antibody [50:1], the ELP remains at great excess to IgM complexes. This
ratio would favor saturation of variable fragments on the IgM, leaving mostly unbound V96
available to phase separate as observed near 37 °C (Figure 17A). To quantify the reproducibility
of this effect, the Area Under the Curve for the OD at 350 nm from 30 to 50 °C was compared
from independent studies (n=3). This confirmed that ratios of V96 to antibody of [50:1] to [5:1]
showed significantly less temperature-dependent change in optical density compared to V96
alone (Figure 17B). Interestingly, when the anti-ELP antibody concentration increased to a molar
ratio of V96 to antibody [5:1], temperature-dependent change in OD was no longer observed.
With less than 10 ELP chains per pentameric IgM complex, there may remain few unbound ELPs
available to phase separate. Loss of phase separation does not appear to require complete
saturation of every ELP binding motif. Even at the lowest ratio of 3:1 per IgM evaluated, there
remains a significant 29:1 excess of ELP binding sites (96 VPGVG repeats x 1 μM = 96 μM) to the
variable fragments on the IgM complex (10 Fv domains x 0.33 μM = 3.3 μM). One explanation
could be that when AK1’s concentration is high enough, ELPs interact at multiple sites with the
same pentameric IgM complex, which constrains ELPs into complex networks unable to
participate in temperature-dependent phase separation (Figure 17C). An irrelevant IgM antibody
47
was evaluated under identical ratios in ELP-mediated phase separation with V96, which yielded
minimal temperature-dependent change in optical density (Figure 18). Since ELPs that phase
separate have been proposed for various therapeutic strategies, the fact that AK1 blocks ELP-
mediated phase separation suggests that an antibody response may change the clearance of both
ELP coacervates as well as soluble ELP therapies.
2.4 Discussion

A novel mAb that is highly reactive against ELPs has been developed in this study. Although
development of a polyclonal antibody would have been less labor intensive, the goal of this study
was to develop a mAb to reduce batch-to-batch variability as much as possible. Moreover,
polyclonal antibodies are more prone to cross reactivity, therefore, a mAb was developed to
eliminate background noise and minimize cross reactivity. Having been derived from human
tropoelastin, ELPs are believed to be weak immunogens because they are greatly conserved and
are abundant in the extracellular matrix of mammalian cells. However, there are various
established techniques that were applied in this study to augment immunogenicity of ELPs.
22
For
example, antigens were emulsified in Freund’s adjuvant. This adjuvants is a water in oil emulsion
that allows prolonged presentation of the antigen to the immune system.
23
In addition, murine
Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used as a priming agent during
the immunization.
24
GM-CSF has the ability to stimulate humoral immunity, resulting in an
enhanced antibody response as well.
25
GM-CSF was used in all injection mixtures during the
immunization except for the last booster injection the animals received. While antigens were co-
injected with the above-mentioned immuno-modulators, antibody titer was measured in the
sera of immunized mice to ensure adequacy of antibody response. The mouse with the highest
48
titer for all three antigens was used for hybridoma production and the positive cell populations
were then subcloned to isolate monoclonal cell lines. Monoclonal antibody-producing cell lines
were further screened and two clones with high reactivity against the three ELPs were selected
for further characterization.
The anti-ELP antibody that was produced in this study was of an IgM isotype as revealed
by an antibody isotyping kit. Reactivity of the antibody was tested against ELPs in an ELISA assay
and was revealed to be high since it was able to detect ELPs even when it was diluted in the low-
nanomolar range. The high reactivity illustrated in the ELISA assay may be related to the
immunomodulators used during the immunization to enhance response to ELP antigens.
Specificity of the antigen-antibody interaction was tested in Western Blotting assays. AK1
was only able to detect ELPs and ELP fusion proteins, but not BSA or other protein structures in
crude cell lysates. Interestingly, the purified AK1 was able to detect a comprehensive library of
ELP and ELP fusion proteins, and specificity was not limited to the three ELPs that were used in
immunization. Furthermore, there was no cross-reactivity with other proteins that did not
contain the pentapeptide of an ELP. For example, a series of cell lysates with or without ELP fusion
proteins were used for immunoblots, and the antibody specifically reacted with ELP fusion
proteins and no other proteins in the lysate. The western blotting studies proved that the
antibody is highly specific for ELP containing constructs regardless of their guest residue, and do
not show unexpected reactivity with other proteins. Lack of AK1 reactivity with proteins in
mammalian cells that were not transfected with an ELP or ELP fusion in immunofluorescence
microscopy experiments further illustrates specificity of these antibodies for ELPs. The finding
49
that the antibody can detect a broad range of ELPs may be due to a common epitope, Gly-Val-
Pro-Gly, found in all of these constructs.
ELPs have gained increased attention over the past decade in a variety of different fields
of study, from drug delivery, to issue engineering, and more.
26-32
Investigators have attempted
various techniques such as epitope tagging for tracking ELPs because of lack of anti-ELP
antibodies.
33, 34
Even though the aforementioned techniques made is easier to study ELPs, access
to anti-ELP mAbs such as AK1 would have been far superior to incorporating epitope tagging. This
is because tags will have immunogenicity of their own, may interfere with protein structure and
stability, and cannot represent the biodegradation products of an entire length of a recombinant
protein-polymer to which they are fused.
In this study, AK1 mAbs were achieved using reagents that increase antigen
immunogenicity; furthermore, these conditions differ greatly in dose/frequency/composition
from potential therapeutic studies of ELPs. This study proves that mice are capable of mounting
an antibody response to aliphatic ELPs, which may even alter their phase behavior. This should
be carefully considered in clinical studies of ELPs because even at low concentrations, an antibody
response to a protein therapy may significantly alter its pharmacokinetic and pharmacodynamic
behavior. Therefore, potential immunogenicity of ELP constructs, and their effect on clinical
outcomes should be investigated at earlier stages.
The anti-ELP mAb, AK1 identified by this study offers considerable potential for future
applications of ELPs, which may advance the clinical development of this protein polymer.
Potential applications now include optimization of bioprocessing, quality control, cellular and
50
tissue localization, toxicology, and ADME (absorption, distribution, metabolism, and elimination)
studies for ELPs and their fusions.
This manuscript presents direct evidence that novel murine mAbs can be raised against
purified ELPs. Due to the breadth of potential ELP applications in the biomedical field, the anti-
ELP AK1 can become an invaluable tool to any research team that is currently studying this class
of biopolymers. The anti-ELP AK1 proved to be highly reactive and specific towards ELPs
regardless of their guest residues in a variety of experiments. Since they bind both ELPs and ELP
fusion proteins, these anti-ELP AK1 antibodies will facilitate purification, experimental detection,
and characterization of these emerging biopolymers.

51
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4. Dhandhukia JP, Brill DA, Kouhi A, Pastuszka MK, MacKay JA. Elastin-like polypeptide
switches: A design strategy to detect multimeric proteins. Protein Sci 2017; 26:1785-95.
5. Despanie J, Dhandhukia JP, Hamm-Alvarez SF, MacKay JA. Elastin-like polypeptides:
Therapeutic applications for an emerging class of nanomedicines. J Control Release 2016;
240:93-108.
6. Janib SM, Pastuszka M, Aluri S, Folchman-Wagner Z, Hsueh PY, Shi P, et al. A
quantitative recipe for engineering protein polymer nanoparticles. Polym Chem 2014; 5:1614-
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7. MacEwan SR, Chilkoti A. Elastin-like polypeptides: biomedical applications of tunable
biopolymers. Biopolymers 2010; 94:60-77.
8. Pastuszka MK, Okamoto CT, Hamm-Alvarez SF, MacKay JA. Flipping the Switch on
Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains. Adv Funct
Mater 2014; 24:5340-7.
9. Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci
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11. Clough NEC, Hauer PJ. Using Polyclonal and Monoclonal Antibodies in Regulatory
Testing of Biological Products. ILAR Journal 2005; 46:300-6.
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reconstruction allows rapid and seamless cloning of oligomeric genes. Biomacromolecules
2010; 11:944-52.
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14. Golemis E, Adams PD. Protein-protein interactions : a molecular cloning manual. Cold
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16. Urry DW. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by
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17. de StGroth SF, Scheidegger D. Production of monoclonal antibodies: strategy and
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18. Janib SM, Liu S, Park R, Pastuszka MK, Shi P, Moses AS, et al. Kinetic quantification of
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19. Meyer DE, Chilkoti A. Purification of recombinant proteins by fusion with thermally-
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peptide-1 fusion protein reduces blood glucose for five days with a single injection. J Control
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22. Leenaars M, Hendriksen CF. Critical steps in the production of polyclonal and
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23. Stills HF, Jr. Adjuvants and antibody production: dispelling the myths associated with
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53
2.7 Figures

Figure 8. Purity and identity of ELPs used in immunization cocktail and/or characterization
assays
Heterologous expression of ELPs was performed in E. coli, expressed through shaker flask
cultures, and then purified through iterative induction of the ELP-mediated phase separation.
Purified ELPs (Table 1) were evaluated for identity and purity using SDS-PAGE stained with
copper chloride.

54

Figure 9. Aliphatic ELPs stain poorly using Coomassie blue on SDS-PAGE.
Supplemental Figure S1. Aliphatic ELPs stain poorly using Coomassie blue on SDS-
PAGE. Heterologous expression of ELPs was performed in E. coli, expressed through
shaker flask cultures, and then purified through iterative induction of the ELP-
mediated phase separation. Purified ELPs (Table 1) were evaluated for identity and
purity using SDS-PAGE stained with Coomassie blue.

55

Figure 10. Phase behavior for ELPs used in combination antigen immunization
Optical density (350 nm) was plotted as a function of concentration and temperature for (a) A192;
(b) A96I96; and (c) S48I48. (d) Concentration–temperature phase diagrams for the bulk phase
transition temperature (Tt,bulk) of A192, A96I96 and S48I48 and the critical micelle temperature
(CMT) of A96I96 and S48I48. The dashed line indicates the 95% confidence interval around a log-
linear fit-line. Based on these phase diagrams, diblock copolymers A96I96 and S48I48 were
intended to assemble nanoparticles at physiological temperatures, while A192 remains soluble.

56

Figure 11. Indirect ELISA of serum titer against purified ELPs was used to assess the
combination immunization of mice
Antibody response was measured in all 5 mice 6-8 weeks after beginning immunization against
purified (a) A192; (b) A96I96; and (c) S48I48. All sera were tested in triplicate, and indicated
errors are the mean ± SD. (n=3). Mice #2 and #5 were selected for subsequent immunization
and hybridoma production based on these ELISA results.
57

Figure 12. Purified anti-ELP AK1 detects a panel of ELPs using indirect ELISA
(a) Immuno-reactivity of AK1 was first tested against the ELPs used in immunization of the
mice. Based fitting to Eq. 1, the apparent Kd for A96I96 is 0.6 nM ± 0.06 nM, for A192 is 0.8
nM ±0.1 nM, and for S48I48 is 0.4 nM ±0.07 nM. (b) Immuno-reactivity of AK1 was then
confirmed against three purified monoblock ELPs, which were not included in the
immunization protocol. The Kd for V96 is 0.6 nM ±0.07 nM, for S96 is 5 nM ±0.4 nM, and for
I48 is 0.6 nM ±0.07 nM. Data and estimates of Kd represent the mean ± SD (n=3).
58

Figure 13. AK1 detects ELPs but not BSA during western immunoblotting.
(a) 0.1 µg of purified ELPs (Table 1) were electrophoresed on lanes 2-4, and 0.1 µg of
BSA (67 kD) was loaded onto lane 5. The sample was detected using AK1 and then using
secondary immunofluorescence as described in the main text. AK1 does not react with
bovine serum albumin (BSA) protein.

59

Figure 14. AK1 efficiently detects ELPs during western immunoblotting from cellular lysates.
(a) 0.1 µg of purified ELPs (Table 1) were electrophoresed on lanes 2-7, while 2 µg was loaded
onto lane 8. To achieve equivalent detection, I48 required a higher loading amount than the
other ELPs, which possibly correlates with its less efficient electro-transfer to the
nitrocellulose membrane. (b) Immunoblotting was also used to verify the expression and
purity of two ELP clathrin-light chain fusions from cell lysates (20 µg) of HEK293T cells. Slightly
higher than their MW, these ELP fusions run near 75 kD. A 37 kD band was identified using an
anti-GAPDH mAb control. The control is untransfected 293T cell lysate. (c) AK1 was used to
track A192 during two rounds of ELP-mediated purification, which suggests that AK1 may be
useful to optimize ELP purification procedures even in complex bacterial lysates. ‘Clarified
lysate’ was separated from the ‘insoluble pellet’ and A192 was purified through two rounds
of ELP-mediated phase separation. The pellet (pel) and supernatant (sup) for hot
centrifugation spins (HS) and cold centrifugation spins (CS) are indicated.

60

Figure 15. Immunofluorescence microscopy using anti-ELP AK1 detects temperature-
dependent assembly of ELP fusions in mammalian cells.
293T cells were transfected with plasmids encoding ELP CLC fusions, pre-chilled at 4 °C,
followed by incubation at 4 or 37 °C and labeled by anti-myc antibody (red), followed by anti-
ELP AK1 (green). When cells expressing both ELP fusions were incubated at 4 °C, proteins
remained diffuse as shown by the staining pattern of both anti-myc and AK1 antibodies.
Consistent with our previous reports, A96-CLC remained soluble even when cells were
incubated at 37 °C, which is below the intracellular T t of A96-CLC. When V96-CLC transfected
cells were incubated at 37 °C, the ELP fusion proteins assembled microdomains as seen in
staining patterns of both anti-myc antibody and anti-ELP AK1.
61

Figure 16. Immunofluorescence microscopy using anti-ELP AK1 detects ELP fusions in
mammalian cells, but does not cross react with other proteins in untransfected cells.
Supplemental Figure S3. Immunofluorescence microscopy using anti-ELP AK1 detects ELP
fusions in mammalian cells, but does not cross react with other proteins in untransfected
cells. 293T cells were either transfected with plasmids encoding ELP CLC fusions or not
transfected with any construct. Cells were then incubated at 4 °C or 37 °C, fixed and stained
by anti-ELP AK1 (green). AK1 antibodies successfully stained cells expressing V96-CLC.
However, when untransfected 293T cells were incubated with AK1, no staining was observed.
This is consistent with the specificity of AK1 for ELPs and ELP fusion proteins.
62

Figure 17. An optimal ratio of anti-ELP AK1 suppresses the ELP temperature-dependent shift
in optical density (OD).
(a) The OD–temperature relationship was collected at 350 nm for V96 mixed with AK1 IgM
antibodies. The V96 concentration was fixed at 1 μM and the AK1 concentration was mixed at
the molar ratios of V96 to antibody of 50:1, 10:1, 5:1, 3:1 and incubated for 30 mins prior to
heating. V96 alone phase separates above 37 °C, which is visible by an increase in OD.
Incubation with a 3:1 or 5:1 ratio of V96 to antibody eliminated the temperature-dependent
increase in OD. (b) The AUC for the OD350 from 30 to 50 °C was determined using Eq. 2 to
quantify the extent of temperature-dependent rise in optical density due to ELP phase
separation. This value was highly dependent on the mixture ratio of V96 to antibody. Indicated
errors are the mean ± SD. (n=3). (Tukey HSD, α=0.05, *p<0.05, **p<0.005, ***p<0.001,
****p<0.0001) (c) For molar ratios of V96 to antibody of 3:1 and 5:1, the elevated baseline
OD may be consistent with the formation of temperature-independent networks interacting
with AK1. For molar ratios of V96 to antibody 50:1 and 10:1, some fraction of ELP remains free
to coacervate at a temperature near 37 °C.

V96
V96:antibody=50:1
V96:antibody=10:1
V96:antibody=5:1
V96:antibody=3:1
-0.4
0.0
0.4
0.8

AUC of OD 350nm
30 to 50 (
o
c )

**
*
**** ****
***
****
****
***
*
35 40 45 50
-0.10
-0.06
-0.02
0.02
0.06
0.10
0.14
0.18
Temperature (
o
C)
OD 350nm
V96
V96:antibody=3:1
V96:antibody=5:1
V96:antibody=10:1
V96:antibody=50:1
a
c
b
V96:antibody=10:1 V96:antibody=5:1
ΔT ΔT ΔT
é coacervate no é coacervate
V96:antibody=50:1
63

Figure 18. NK-1 antibody of IgM isotype does not suppress the ELP temperature-dependent
shift in optical density (OD)
Supplemental Figure S4. NK-1 antibody of IgM isotype does not suppress the ELP
temperature-dependent shift in optical density (OD). The OD–temperature relationship was
collected at 350 nm for V96 mixed with an NK-1 IgM antibody, which binds an antigen
irrelevant to ELPs. The V96 concentration was fixed at 1 μM and the NK-1 antibody
concentration was mixed at the molar ratios of V96:IgM of 50:1, 10:1, 5:1, 3:1 and incubated
for 30 min prior to heating. V96 alone phase separates above 37 °C, which is visible by an
increase in OD. Incubation with an IgM antibody that doesn’t bind ELPs does not suppress the
transition temperature of ELPs.

35 40 45 50
-0.10
-0.06
-0.02
0.02
0.06
0.10
0.14
0.18
Temperature (
o
C)
OD 350nm
V96
V96:antibody=3:1
V96:antibody=5:1
V96:antibody=10:1
V96:antibody=50:1
64

Figure 19. Plasmid map of ELP constructs inside pet25b(+)backbone.
Recombinant pET-25b(+) plasmids, containing ELP genes, were previously engineered in our
lab using recursive directional ligation method.

(8089) AcuI
BssHII (3964)
BssHII (3964)
(8089) AcuI
A192 ELP inside pET-25b(+) backbone A96I96 ELP inside pET-25b(+) backbone
BssHII (3964)
(720) AcuI
S96 ELP inside pET-25b(+) backbone
(5929) AcuI
I48 ELP inside pET-25b(+) backbone
BssHII (3964)
(720) AcuI
V96 ELP inside pET-25b(+) backbone
(6649) AcuI
S48I48 ELP inside pET-25b(+) backbone
65

2.8 Tables

Table 2.1. Library of ELPs and ELP fusion proteins used in this study
ELP
Nomenclature
Amino Acid Sequence
Tt, bulk , CMT
(°C)
MW
g

(kD)
MW
h

(kD)
Purity
j
(%)
A96I96
a
MG(VPGAG) 96(VPGIG) 96Y 46.5, 19.2
c
77.7 77.8
i
94.3
A192
a
MG(VPGAG) 192Y 59.8, na
c
73.6 73.2 96.8
S48I48
a
MG(VPGSG) 48(VPGIG) 48Y 73.7, 23.5
c
39.8 39.7 97.8
V96 MG(VPGVG) 96Y 31.9, na
d
39.7 39.7 93.2
S96 MG(VPGSG) 96Y 62.6, na
d
38.5 38.3 97.2
I48 MG(VPGIG) 48Y 31, na
e
20.7 20.7 90.1
A96-CLC MGLGG(VPGAG) 96-myc-CLC
b
>42
f
62.3 na na
V96-CLC MGLGG(VPGVG) 96-myc-CLC
b
37
f
65.0 na na
a
The three ELPs that were used in combination immunization of the mice.
b
myc-CLC represents a myc antigen followed by the clathrin light chain sequence as
follows:RLEEQKLISEEDLLEMAELDPFGAPAGAPGGPALGNGVAGAGEEDPAAAFLAQ
QESEIAGIENDEAFAILDGGAPGPQPHGEPPGGPDAVDGVMNGEYYQESNGPTDSYAA
ISQVDRLQSEPESIRKWREEQMERLEALDANSRKQEAEWKEKAIKELEEWYARQDEQL
QKTKANNRAAEEAFVNDIDESSPGTEWERVARLCDFNPKSSKQAKDVSRMRSVLISLK
QAPLV
c
The Tt,bulk and CMT where determined from optical density (350 nm) of a 25 μM
polypeptide in PBS, na (not applicable)
d
Tt estimated by Shah and coworkers
2
, na (not applicable)
e
Tt estimated by Janib and coworkers
6
, na (not applicable)

f
intracellular Tt estimated by Pastuszka and coworkers
8

g
estimated from DNA sequence of expressed ELP
h
confirmed using MALDI-TOF, na (not applicable)
i
confirmed using MALDI-TOF in prior publication by Janib and coworkers
6
j
estimated from SDS-PAGE of purified ELPs evaluated in this work, na (not applicable)
66
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Abstract (if available)

Abstract The first chapter of this dissertation includes work on development of an immunotherapy for treatment of ovarian cancer. Anti-HER2 CAR T-cells were developed to treat ovarian cancer solid tumors that express HER2. CAR constructs were generated by fusing anti-HER2 scFv to a second-generation CAR. The CAR vector was then fused with a lentivirus vector in-frame with the CAR backbone and was used to transduce primary human CD3 positive T-cells. After transduction, expanded CAR-T cells were characterized to confirm their ability to bind HER-2 antigen on HER-2 positive SK-OV-3 cells using flow cytometry. Expanded CAR-T cells were then tested for their epitope-driven cytotoxicity by co-culturing them with HER-2 positive tumor cells. In this assay, HER-2 CAR T-cells displayed dose-dependent cytotoxicity when co-cultured with antigen positive but not antigen negative tumor cells. HER2 CAR T-cells were then tested in vivo in an in-house intraperitoneal, metastatic model of human ovarian cancer that is used for testing the efficacy of CAR-T cells against intraperitoneal tumors. Using this SK-OV-3/NSG mouse model, HER-2 CAR T-cells were found to reduce in a significant manner tumor burden, while tumor progression continued in mice that were treated with the controls. Results of this study show that HER-2 CAR T cells have antigen driven effector functions both in vivo and in vitro and can complement the current standard of care as a targeted therapy to improve the prognosis of HER2 expressing ovarian cancer patients. In order to translate this product to the clinic, the murine HER-2 antibody is presently being out-sourced for humanization. ? The second chapter, which is a study that is published focuses on development of antibodies against an emerging class of peptide biologics is known as the Elastin-like polypeptide (ELP). The identification and use of antibodies dominate the biologic, clinical diagnostic, and therapeutic landscapes. In particular, antibodies have become essential tools in a variety of protein analytical experiments and to study the disposition of biologic therapeutics. ELPs are repetitive protein polymers inspired by human tropoelastin. A major limitation in the clinical translation of ELP biologics has been a lack of a monoclonal antibody (mAb) to characterize their identity during expression. To facilitate these studies, we successfully generated a new mAb that is specific towards ELPs and ELP fusion proteins. Purified antibody was evaluated in ELISA, Western Blotting, and immunofluorescence assays for their analytical potential. The optimal anti-ELP mAb proved highly reactive and specific towards ELPs. Moreover, these novel antibodies were able to detect ELPs with a variety of aliphatic guest residues. ELPs phase separate in response to heating. When incubated at great excess to ELP, the anti-ELP mAb partially blocks phase separation. These findings are direct evidence that these novel murine mAbs will enable purification, experimental detection, and characterization of these useful biopolymers.

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Asset Metadata

Creator Kouhi, Aida (author)

Core Title Immunotherapy of cancer

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/23/2023

Defense Date 12/22/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag CAR-T cell therapy,elastin-like polypeptide,immunotherapy of cancer,monoclonal antibody development,OAI-PMH Harvest,ovarian cancer

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Epstein, Alan Lee (committee chair), Kaslow, Harvey (committee member), Okamoto, Curtis (committee member)

Creator Email aidakouhi@gmail.com,kouhiaida@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15623626

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